Methods and apparatus for sterility testing

ABSTRACT

Methods and apparatus for testing the sterility of a product to be administered to a patient. The sterility of the product is tested by monitoring the pH change of a culture medium in contact with any culturable organisms (e.g., bacteria) that are collected by filtration of the product to be tested.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/059,690, filed Jun. 6, 2008, and is a continuation-in-part of U.S.patent application Ser. No. 11/207,580, filed Aug. 19, 2005. Eachapplication is expressly incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to methods and apparatus for testing thesterility of a product to be administered to a patient.

BACKGROUND OF THE INVENTION

Platelets are a component of blood comprised of anucleate megakaryocytefragments that circulate in the blood for about 10 days (van der Meer,Pietersz et al. 2001; Dijkstra-Tiekstra 2004). As they age in thecirculatory system platelets are known to undergo biochemical changesthat eventually leads to their clearance in the spleen and liver. Whenseparated as a component of whole blood, platelets are routinelyconcentrated, resuspended in plasma and/or platelet additive solutions,leukoreduced by passage through a filtration device and stored inplatelet storage bags which are kept on flatbed agitators for 5 to 7days at a temperature of 22° C.

The measurement of pH and other parameters during preparation andstorage of blood components are necessary in order to provide a safe andeffective product. For example the storage of platelets at 22° C.requires testing for the presence of microbiological contamination toprevent undesired side effects such as sepsis as a result of infusioninto the patient. Under the American Association of Blood Banks(A.A.B.B.) standard 5.1.5.1, blood banks or transfusion services areinstructed to have methods to limit and detect bacterial contaminationin all platelets. The growth of bacteria in platelet concentrates (PCs)can be monitored by utilizing reagent dipsticks for pH and glucosehowever the time of detection following inoculation with a low dose oforganisms (e.g. 50 colony forming units/mL) varies from organism toorganism, limiting this method's sensitivity and specificity given theshelf life of the product (Brecher, Hogan et al. 1994). Even in light ofthese limitations many centers have moved to time of issue testsutilizing the measurement of pH and glucose with a handheld device, pHpaper, or in combination on a multi-reagent dipstick as surrogatemarkers for bacterial contamination (Burstain, Brecher et al. 1997;Yazer and Triulzi 2005).

In one method of non-invasive bacterial detection, changes in pH or theproduction of CO₂ was detected in clinical specimens by culturing thespecimens with a sterile liquid growth medium in a transparent sealedcontainer (Calandra et al., U.S. Pat. No. 5,094,955). The maindisadvantage of this method is the requirement for sampling of aclinical specimen and introduction of the sample into a separate culturevessel at one point in time. The sampling is disadvantageous because thecontaminating organisms may not be included in the sample volume,resulting in a false negative test. A similar method employed a sensorto detect microbial organisms growing in a liquid environment with themicrobial colonies immediately available for further testing by virtueof the design of the culture vessel (Jeffrey et al., U.S. Pat. No.5,976,827). This general method by which culture-based bacterialdetection systems function is currently used for detection ofmicroorganisms in PCs (Brecher, Means et al. 2001; McDonald, Pearce etal. 2005). A device for measuring the pH of PCs non-invasively by afluorescence-based interrogation of the bag contents has been describedin the WO 2006/023725 (PCT/US2005/029559), expressly incorporated hereinby reference in its entirety.

Additional important reasons to measure pH in the quality control of PCsinclude its correlation with in vivo viability following transfusioninto patients (Moroff, Friedman et al. 1982; Solberg, Holme et al. 1986;Holme 1998; Rinder, Snyder et al. 2003). pH Values below 6.2 in PCs havebeen correlated with poor in vivo recovery in transfusion studies(Murphy and Gardner 1971; Slichter and Harker 1976; Murphy 1985), whileloss of recovery in vivo at pH values above 7.2 has been shown (Murphyand Gardner 1975). The platelet yield in PCs is also an importantquality control parameter because it establishes the therapeutic dosageof the product and may influence the levels of metabolic activitymeasured within the storage bags. There have been several studiesshowing maintenance of pH values indicative of good platelet function upto 7 or 8 days in PCs stored in mixtures of additive solutions andplasma (Klinger 1996), provided that the platelet content was <4×10¹¹(deWildt-Eggen, Schrijver et al. 1998). These latter studies showed that amore rapid decline in pH in a PC may correlate with higher plateletconcentrations. A similar correlation of a more rapid decline in pH overthe five day storage period was inferred in apheresis derived PCsobtained from machines that consistently produce product with higherplatelet counts (Tudisco, Jett et al. 2005).

The storage of platelets in BTHC (n-butyryl, tri-n-hexyl citrate) PVCcontainers presents a number of advantages with respect to platelethealth. These containers have several desirable characteristics for thismedical application including low toxicity and permeability to water,O₂, and CO₂ in the desired ranges. The BTHC plasticized PVC materialalso has high permeability for CO₂, excess of which in some cases leadsto difficulties storing PCs which have high platelet counts. Dependingon storage conditions the pH of PCs can change rapidly due tooff-gassing of dissolved CO₂. The Council of Europe guidelines forplatelet storage conditions require the pH to be in the range of 6.4-7.4at 37° C. (6.6-7.6 at 22° C.).

Optical sensors (optrodes) for measuring pH are well known. Certainaromatic organic compounds (like phenolphthalein) change color with pHand can be immobilized on solid supports to form “pH paper.” Thesevisual indicators are easy to use, but do not provide a quantitativereading. The color changes can be difficult to distinguish accurately,and can be masked by colored analyte. Fluorescent indicators have alsobeen used as optical sensors. pH Sensitive fluorescent dyes can beimmobilized on solid supports and generally are more sensitive incomparison to the simple colorimetric (absorbance or reflectance based)indicators. The improved sensitivity of fluorescent indicators allowsthe solid support to be miniaturized, and this has been used toadvantage in development of fiber optic sensor devices for measuring pH,CO₂, and O₂ parameters in blood.

A specific need in the medical industry exists for accurate pHmeasurement of blood and blood products. The pH of blood or other bodilyfluids (pleural effusions) can be associated with certain physiologicresponses associated with pathology. Blood gas analyzers are commoncritical care instruments. Depending on storage conditions, the pH ofseparated blood components (plasma, platelets) can change rapidly due tooff-gassing of dissolved CO₂ from the enriched venous blood that iscollected from a donor. Platelets in particular are metabolicallyactive, and generate lactic acid during storage at 20° C. to 22° C.European quality guidelines for platelets prepared by the “buffycoatmethod” require pH of stored platelets to be pH 6.8-7.4 at 37° C.(7.0-7.6 at 22° C.).

Seminaphthofluorescein (SNAFL) compounds and the relatedseminaphthorhodafluor (SNARF) compounds are commercially availableratiometric fluors (Molecular Probes, Inc., Eugene, Oreg.; see, forexample, U.S. Pat. No. 4,945,171) and their synthesis and spectralproperties have been described. These compounds have advantagesincluding long wavelength absorbance that can be efficiently excitedwith LED light sources. Relevant acid/base equilibria and associatedspectral properties are shown below.

Deprotonation of the naphthol structure of SNAFL dyes gives anaphtholate molecule with longer wavelength fluorescence emission. ThepKa is the pH value where the two molecular species form in equalamounts. SNAFL compounds with reactive linker groups that allow theirconjugation to other molecules of interest are also commerciallyavailable.

Various methods have been used to immobilize “ratiometric” dyes to solidsupports for use in fiber optic pH detectors. Carboxynaphthofluorescein(CNF) has been conjugated to aminoethyl-cellulose and this material wasglued to polyester (Mylar) films to make sensing membranes for optrodes.The pKa of this material was determined to be 7.41, slightly lower thanthe free CNF (pKa 7.62). The use of tetraethoxysilane to trap CNF in asol-gel glass that was formed on glass cover slips has also beenreported. The pKa of this material was determined to be 7.46. A 9-chlorosubstituted SNAFL analog (SNAFL-2) has been reacted with polyvinylamineand the residual amino groups crosslinked with a photocrosslinker toform a gel-like coating on acrylic fibers. The pKa of this fiber-opticsensor was determined to be 7.14, significantly lower than the publishedpKa of the free SNAFL compound (pKa ˜7.7). This shows that molecularenvironment and linker structure surrounding the immobilized dye canalter the performance of a pH detector.

Despite the advances made in the detection of pH noted above, thereexists a need for improved methods and devices for monitoring thechemical environment in a sealed sterile container, continually or atdiscrete time intervals, in order to better understand the types andlevels of metabolic activities within the container, as well as theirorigin. The present invention seeks to fulfill this need and providesfurther related advantages.

SUMMARY OF THE INVENTION

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This summary is not intended to identify key features ofthe claimed subject matter, nor is it intended to be used as an aid indetermining the scope of the claimed subject matter.

In one aspect, the invention provides a method for testing the sterilityof a sample. In one embodiment, the method includes:

(a) providing a filter device having a chamber and a filter configuredto collect culturable organisms;

(b) filtering a sample through the filter such that any collectedculturable organisms are retained in the chamber;

(c) adding a culture medium to the chamber;

(d) irradiating a fluorescent species immobilized on a substrate withexcitation light emanating from a probe physically isolated from thefluorescent species immobilized on the substrate,

wherein the fluorescent species immobilized on the substrate is inliquid communication with the culture medium,

wherein the excitation light has a wavelength sufficient to effectfluorescent emission from the fluorescent species, and

wherein the fluorescent species exhibits a first emission intensity at afirst emission wavelength and a second emission intensity at a secondemission wavelength, the ratio of the first and second emissionintensities being dependent on pH;

(e) measuring the first and second emission intensities to determine thepH of the culture medium; and

(f) inferring from the determined pH of the culture medium the sterilityof the sample.

In one embodiment, the filter includes a membrane having a pore size ofabout 45 μm.

In one embodiment, the probe is physically isolated from the fluorescentspecies immobilized on the substrate by a window transparent to theexcitation light and the fluorescent emission.

In one embodiment, the probe includes one or more optical fibers.

In one embodiment, the fluorescent species is selected from the groupconsisting of a naphthofluorescein compound and a seminaphthorhodaminecompound.

In one embodiment, the fluorescent species is a naphthofluoresceincompound selected from the group consisting of a seminaphthofluoresceincompound and a carboxynaphthofluorescein compound.

In one embodiment, the fluorescent species is a seminaphthofluoresceincompound selected from the group consisting of 5′(and6′)carboxy-3,10-dihydroxy-spiro[7H-benzo[c]xanthene-7,1′(3′H)-isobenzofuran]-3′-oneand2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoicacid.

In one embodiment, the fluorescent species is a conjugate of afluorescent compound and a macromolecule.

In one embodiment, the macromolecule is an albumin.

In one embodiment, the fluorescent species is aseminaphthofluorescein/human serum albumin conjugate.

In one embodiment, the sample is blood or a blood product.

In another aspect, the invention provides a method for testing thesterility of a sample. In one embodiment, the method includes:

(a) providing a filter device having a chamber and a filter configuredto collect culturable organisms;

(b) filtering a sample through the filter such that any collectedculturable organisms are retained in the chamber;

(c) adding a culture medium to the chamber;

(d) irradiating a fluorescent species in communication with the culturemedium with excitation light having a wavelength sufficient to effectfluorescent emission from the fluorescent species,

wherein the fluorescent species exhibits a first emission intensity at afirst emission wavelength and a second emission intensity at a secondemission wavelength, the ratio of the first and second emissionintensities being dependent on pH, and

wherein the fluorescent species is a 2-halo seminaphthofluorescein;

(e) measuring the first and second emission intensities to determine thepH of the culture medium; and

(f) inferring from the determined pH of the culture medium the sterilityof the sample.

In one embodiment, the 2-halo seminaphthofluorescein is a 2 chloroseminaphthofluorescein.

In one embodiment, the 2-halo seminaphthofluorescein is2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoicacid.

In one embodiment, the fluorescent species is a conjugate of a 2-haloseminaphthofluorescein and a macromolecule.

In one embodiment, the macromolecule is an albumin.

In one embodiment, the macromolecule is a human serum albumin.

In one embodiment, the conjugate is a2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoicacid/human serum albumin conjugate.

In one embodiment, the sample is blood or a blood product.

In another aspect, the invention provides an apparatus for testing thesterility of a sample liquid. In one embodiment, the apparatus includes:

(a) a chamber having an inlet and an outlet;

(b) a filter disposed at the chamber outlet, the filter adapted tocollect culturable organisms from a liquid passed through the filter;and

(c) a port assembly having a distal end extending into the chamber, thedistal end having a fluorescent species immobilized on a substrate,

wherein the fluorescent species is in fluid communication with aninterior of the chamber,

wherein the fluorescent species is a material that, when irradiated withan excitation light having a wavelength sufficient to effect fluorescentemission from the fluorescent species, exhibits a first emissionintensity at a first emission wavelength and a second emission intensityat a second emission wavelength, the ratio of the first and secondemission intensities being dependant on pH, and

wherein the port assembly further includes a housing for receiving aprobe.

In one embodiment, the filter includes a membrane having a pore size ofabout 45 μm.

In one embodiment, the housing comprises an open first end and a secondend terminating with a window.

In one embodiment, the port assembly includes a tip member reversiblyconnectable to the housing second end, wherein the tip member is adaptedto receive liquid from a sample to be tested.

In one embodiment, the apparatus includes:

(a) a light source for exciting the fluorescent species;

(b) a first emission detector for measuring the first emissionintensity;

(c) a second emission detector for measuring the second emissionintensity;

(d) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the excitationlightguide has a first terminus proximate to the light source and asecond terminus distal to the light source;

(e) a first emission lightguide for transmitting emission from thefluorescent species to the first emission detector, wherein the firstemission lightguide has a first terminus proximate to the first emissiondetector and a second terminus;

(f) a second emission lightguide for transmitting emission from thefluorescent species to the second emission detector, wherein the secondemission lightguide has a first terminus proximate to the secondemission detector and a second terminus; and

(g) a probe housing, wherein the probe housing houses the second terminiof the excitation lightguide, first emission lightguide, and secondemission lightguide.

In one embodiment, the light source is a light-emitting diode.

In one embodiment, the first and second emission detectors arephotodiodes.

In one embodiment, the excitation lightguide, the first emissionlightguide, and the second emission lightguide are optical fibers.

In one embodiment, the port assembly includes a tapered tube terminatingwith the window.

In one embodiment, the fluorescent species is aseminaphthofluorescein/human serum albumin conjugate.

In another aspect, the invention provides an apparatus for testing thesterility of a liquid sample. In one embodiment, the apparatus includes:

(a) a chamber having an inlet and an outlet;

(b) a filter disposed at the chamber outlet, the filter adapted tocollect culturable organisms from a liquid passed through the filter,the filter having a fluorescent species immobilized thereon,

wherein the fluorescent species is in fluid communication with aninterior of the chamber,

wherein the fluorescent species is a material that, when irradiated withan excitation light having a wavelength sufficient to effect fluorescentemission from the fluorescent species, exhibits a first emissionintensity at a first emission wavelength and a second emission intensityat a second emission wavelength, the ratio of the first and secondemission intensities being dependant on pH; and

(c) a port assembly having a distal end adjacent to the filter, the portassembly further including a housing for receiving a probe.

In one embodiment, the filter includes a membrane having a pore size ofabout 45 μm.

In one embodiment, the housing comprises an open first end and a secondend terminating with a window.

In one embodiment, the apparatus includes:

(a) a light source for exciting the fluorescent species;

(b) a first emission detector for measuring the first emissionintensity;

(c) a second emission detector for measuring the second emissionintensity;

(d) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the excitationlightguide has a first terminus proximate to the light source and asecond terminus distal to the light source;

(e) a first emission lightguide for transmitting emission from thefluorescent species to the first emission detector, wherein the firstemission lightguide has a first terminus proximate to the first emissiondetector and a second terminus;

(f) a second emission lightguide for transmitting emission from thefluorescent species to the second emission detector, wherein the secondemission lightguide has a first terminus proximate to the secondemission detector and a second terminus; and

(g) a probe housing, wherein the probe housing houses the second terminiof the excitation lightguide, first emission lightguide, and secondemission lightguide.

In one embodiment, the light source is a light-emitting diode.

In one embodiment, the first and second emission detectors arephotodiodes.

In one embodiment, the excitation lightguide, the first emissionlightguide, and the second emission lightguide are optical fibers.

In one embodiment, the port assembly includes a tapered tube terminatingwith the window.

In one embodiment, the fluorescent species is aseminaphthofluorescein/human serum albumin conjugate.

In another aspect the invention provides a system for testing thesterility of a liquid sample. In one embodiment, the system includes:

(a) a plurality of stations, each station configured to receive aculture assembly;

(b) a plurality of culture assemblies, each culture assembly including:

-   -   (i) a chamber having an inlet and an outlet;    -   (ii) a filter disposed at the chamber outlet, the filter adapted        to collect culturable organisms from a liquid passed through the        filter;    -   (iii) a port assembly having a distal end extending into the        chamber, the distal end having a fluorescent species immobilized        on a substrate,

wherein the fluorescent species is in fluid communication with aninterior of the chamber,

wherein the fluorescent species is a material that, when irradiated withan excitation light having a wavelength sufficient to effect fluorescentemission from the fluorescent species, exhibits a first emissionintensity at a first emission wavelength and a second emission intensityat a second emission wavelength, the ratio of the first and secondemission intensities being dependant on pH; and

wherein the port assembly further includes a housing for receiving aprobe.

In one embodiment, each culture assembly includes:

(a) a light source for exciting the fluorescent species;

(b) a first emission detector for measuring the first emissionintensity;

(c) a second emission detector for measuring the second emissionintensity;

(d) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the excitationlightguide has a first terminus proximate to the light source and asecond terminus distal to the light source;

(e) a first emission lightguide for transmitting emission from thefluorescent species to the first emission detector, wherein the firstemission lightguide has a first terminus proximate to the first emissiondetector and a second terminus;

(f) a second emission lightguide for transmitting emission from thefluorescent species to the second emission detector, wherein the secondemission lightguide has a first terminus proximate to the secondemission detector and a second terminus; and

(g) a probe housing, wherein the probe housing houses the second terminiof the excitation lightguide, first emission lightguide, and secondemission lightguide.

In another aspect, the invention provides a system for testing thesterility of a liquid sample. In one embodiment, the system includes:

(a) a plurality of stations, each station configured to receive aculture assembly;

(b) a plurality of culture assemblies, each culture assembly including:

-   -   (i) a chamber having an inlet and an outlet;    -   (ii) a filter disposed at the chamber outlet, the filter adapted        to collect culturable organisms from a liquid passed through the        filter, the filter having a fluorescent species immobilized        thereon,

wherein the fluorescent species is in fluid communication with aninterior of the chamber,

wherein the fluorescent species is a material that, when irradiated withan excitation light having a wavelength sufficient to effect fluorescentemission from the fluorescent species, exhibits a first emissionintensity at a first emission wavelength and a second emission intensityat a second emission wavelength, the ratio of the first and secondemission intensities being dependant on pH; and

-   -   (iii) a port assembly having a distal end adjacent to the        filter, the port assembly further including a housing for        receiving a probe.

In one embodiment, each culture assembly includes:

(a) a light source for exciting the fluorescent species;

(b) a first emission detector for measuring the first emissionintensity;

(c) a second emission detector for measuring the second emissionintensity;

(d) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the excitationlightguide has a first terminus proximate to the light source and asecond terminus distal to the light source;

(e) a first emission lightguide for transmitting emission from thefluorescent species to the first emission detector, wherein the firstemission lightguide has a first terminus proximate to the first emissiondetector and a second terminus;

(f) a second emission lightguide for transmitting emission from thefluorescent species to the second emission detector, wherein the secondemission lightguide has a first terminus proximate to the secondemission detector and a second terminus; and

(g) a probe housing, wherein the probe housing houses the distal terminiof the excitation lightguide, first emission lightguide, and secondemission lightguide.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 illustrates a large platelet storage bag incorporating a pHreading insert;

FIG. 2 illustrates a small platelet storage bag incorporating a pHreading insert;

FIGS. 3A-3D are graphs comparing pH profiles from different pHmeasurement systems for PC samples stored in platelet storage bags;

FIG. 4A is a graph comparing Kunicki Scores for platelets stored indifferent types of platelet storage bags over a period of time;

FIG. 4B is a graph comparing Swirling Scores for platelets stored indifferent types of platelet storage bags over a period of time;

FIG. 5A is a graph comparing pH profiles of platelets stored indifferent types of platelet storage bags;

FIG. 5B is a graph comparing glucose and lactate concentration profilesof platelets stored in different types of platelet storage bags;

FIG. 6A is a graph comparing ATP:ADP ratio profiles for platelets storedin different types of platelet storage bags;

FIG. 6B is a graph comparing mitochondrial potential profiles forplatelets stored in different types of platelet storage bags;

FIG. 7 is a graph comparing the level of platelet activation measured byCD62P and Annexin V percentages for platelets stored in different typesof platelet storage bags;

FIG. 8 is a graph comparing pH profiles for under-filled andnormal-filled platelet storage bags;

FIGS. 9A-9D are graphs illustrating pH profiles for platelet samplescontaminated with different types of bacteria;

FIGS. 10A-10D are graphs illustrating the pH, glucose, pO₂, and pCO₂profiles, respectively, of platelet samples contaminated with bacteria.

FIG. 11 illustrates a representative multi-probe platelet storage bag;

FIG. 12 is a schematic illustration of an automated single reader;

FIG. 13 is a schematic illustration of a multi-fiber reader;

FIG. 14 is a schematic illustration of an incubator;

FIG. 15 is a graph relating fluorescent intensity ratio to pH of PCs;

FIG. 16 is a graph relating fluorescence intensity ratios to blood gaspH of PCs;

FIG. 17 is a graph comparing pH profiles of various types of plateletsamples.

FIG. 18 is a schematic illustration of a system for measuring pH;

FIG. 19 is a schematic illustration of an optical platform for measuringpH;

FIG. 20 is a schematic illustration of a housing for excitation andemission lightguides;

FIG. 21 illustrates the relationship between the excitation/emissionoptical fiber housing and the sealed vessel port;

FIG. 22 is a port assembly useful in the manufacture of a sealed vessel;

FIGS. 23A-23E illustrate the structures of seminaphthofluoresceincompounds;

FIG. 24 illustrates the emission spectra as a function of pH of afluorescent species (SNAFL-1);

FIG. 25 illustrates the emission spectra as a function of pH of afluorescent species (EBIO-3);

FIG. 26 is a schematic illustration of the preparation of afluorophore-protein (EBIO-3/HSA) conjugate;

FIG. 27 illustrates the emission spectra as a function of pH of afluorophore-protein conjugate (SNAFL-1/HSA);

FIG. 28 illustrates the emission spectra as a function of pH of afluorophore-protein conjugate (EBIO-3/HSA);

FIG. 29 illustrates the emission spectra of a substrate-immobilizedfluorophore-protein conjugate (SNAFL-1/HSA) as a function of pH(Oxyphen);

FIG. 30 illustrates the emission spectra of a substrate-immobilizedfluorophore-protein conjugate (SNAFL-1/HSA) as a function of pH(nitrocellulose);

FIG. 31 illustrates the emission spectra of a substrate-immobilizedfluorophore-protein conjugate (EBIO-3/HSA) as a function of pH(nitrocellulose);

FIG. 32 illustrates the data used in the method for measuring pH;

FIG. 33 illustrates the results of the method for platelet rich plasma;

FIG. 34 illustrates the correlation of pH results for platelet richplasma;

FIG. 35 illustrates stability of a substrate-immobilized fluorophoreconjugate;

FIG. 36 illustrates a device for measuring carbon dioxide in a sealedvessel;

FIG. 37 illustrates the effect of probe position on fluorescentintensity in measuring pH;

FIG. 38 illustrates the effect of membrane pore size on fluorescentintensity in measuring pH;

FIGS. 39A and 39B illustrate representative membrane filter devices ofthe invention;

FIGS. 40A and 40B illustrate other representative membrane filterdevices of the invention;

FIG. 41 is a cross-sectional view of a membrane filter device of theinvention illustrated in FIG. 40A installed in a representative deviceof the invention having a plurality of testing stations;

FIG. 42 illustrates a representative device of the invention having aplurality of stations for receiving filter devices, each stationproviding a probe for interrogating culture medium in the filter device;

FIG. 43 illustrates another representative device of the inventionhaving a plurality of stations for receiving filter devices, eachstation providing a probe for interrogating culture medium in the filterdevice;

FIG. 44 is a cross-sectional view of the representative device of theinvention having a plurality of stations for receiving filter devicesillustrated in FIG. 43; and

FIG. 45 illustrates another representative membrane filter device of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method and apparatus for testingsterility of a sample by detecting and measuring chemical perturbationsin a culture medium containing any culturable organisms collected fromthe sample. Perturbations in the culture medium are detected andmeasured by monitoring the culture medium for changes in a parametermeasurable by the method and apparatus. Parameters measurable by themethod and apparatus include pH and carbon dioxide (CO₂) level. Themethod and apparatus of the invention are useful for detecting bacterialcontamination in a blood sample or a blood product sample.

In one aspect, the a fluorescence method for monitoring a parameter in asample is provided. In one embodiment, the method includes the steps of:

(a) irradiating a fluorescent species having an emission that isdependent on a parameter with excitation light emanating from a probephysically isolated from the fluorescent species to provide afluorescent emission, wherein the fluorescent species is in liquidcommunication with a sample contained in a vessel;

(b) measuring the emission to determine a first parameter reading of thesample; and

(c) repeating step (a) after a pre-determined time and measuring theemission intensity to determine a second parameter reading of thesample.

In one embodiment, step (c) is repeated to provide multiple parameterreadings over time to monitor the parameter of the sample. In oneembodiment, the parameter is pH. In another embodiment, the parameter isCO₂.

In one embodiment, the method further includes writing parameter data tothe vessel containing the sample, the vessel having a means forreceiving the parameter data that further allows the data to be readfrom the vessel at a later time.

In another aspect, a system for monitoring a parameter is provided. Inone embodiment, the system includes:

(a) a light source for exciting a fluorescent species having an emissionthat is dependent on the parameter, wherein the fluorescent species isin liquid communication with a sample contained in a vessel;

(b) an emission detector for measuring emission from the fluorescentspecies and creating emission data;

(c) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the lightguidecomprises a first terminus proximate to the light source and a secondterminus distal to the light source;

(d) an emission lightguide for transmitting emission from thefluorescent species to the emission detector, wherein the lightguidecomprises a first terminus proximate to the detector and a secondterminus distal to the detector;

(e) a probe housing the distal termini of the excitation lightguide andthe emission lightguide;

(f) a housing for receiving the probe, wherein the housing is adaptedfor receiving the probe at a first end and terminating with a window atthe second end, the window being transparent to the excitation and theemission light, and wherein the window physically isolates the probefrom the fluorescent species;

(g) a memory device for storing emission data; and

(h) a processor device for converting the emission data to profile ofthe parameter over time.

In one embodiment, the system further includes a means for writingparameter data to the vessel containing the sample, the vessel having ameans for receiving the parameter data that further allows the data tobe read from the vessel at a later time.

In another aspect, a fluorescence method for monitoring the pH of asample is provided. In one embodiment, the method includes the steps of:

(a) irradiating a fluorescent species in liquid communication with asample contained in a vessel with excitation light emanating from aprobe physically isolated from the fluorescent species, wherein theexcitation light has a wavelength sufficient to effect fluorescentemission from the fluorescent species, wherein the fluorescent speciesexhibits a first emission intensity at a first emission wavelength and asecond emission intensity at a second emission wavelength, the ratio ofthe first and second emission intensities being dependent on pH;

(b) measuring the first and second emission intensities to determine thepH of the sample; and

(c) repeating step (a) after a pre-determined time and measuring thefirst and second emission intensities to determine the second pH of thesample.

In one embodiment, step (c) is repeated to provide multiple pHdeterminations to monitor the pH of the sample over time.

In one embodiment, the method further includes writing pH data to thevessel containing the sample, the vessel having a means for receivingthe parameter data that further allows the data to be read from thevessel at a later time.

In the above methods, the parameter profile or pH profile is made bymultiple parameter or pH measurements. The multiple measurements aremade over time and establish the profile. When the interval of timebetween measurements is sufficiently small, the profile revealsperturbations, if any, in the sample over the time period of theprofile. In the methods, the interval of time between measurements ispredetermined. The pre-determined times are set to reveal perturbationsin the sample.

In one embodiment, the pre-determined time is a variable time periodfrom 1 minute to 1 day. In one embodiment, the pre-determined time isfrom between 1 hour to 12 hours.

In the methods, the multiple parameter or pH determinations over timeprovide a parameter or pH profile of a sample. The parameter or pHprofile of the sample can be used to determine a quality of the sample.

In the above methods, the probe is physically isolated from thefluorescent species. As used herein, the term “physically isolated”refers to the physical isolation of the probe from the sample beinginterrogated. The probe providing excitation light and receivingemission light does contact the sample being interrogated. In themethods, the sample is in contact (i.e., liquid communication) with thefluorescent species (e.g., substrate-immobilized fluorescent species).The probe is isolated from and does not come not physical contact withthe sample. The isolation of the probe from the sample is illustrated inFIG. 21. The probe is isolated from the fluorescent species by a windowtransparent to the excitation light and the fluorescent emission.

In another aspect, a system for monitoring pH is provided. In oneembodiment, the system includes:

(a) a light source for exciting a fluorescent species, wherein thefluorescent species has a first emission intensity at a first emissionwavelength and a second emission intensity at a second emissionwavelength;

(b) a first emission detector for measuring the first emissionintensity;

(c) a second emission detector for measuring the second emissionintensity;

(d) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the lightguidecomprises a first terminus proximate to the light source and a secondterminus distal to the light source;

(e) a first emission lightguide for transmitting emission from thefluorescent species to the first emission detector, wherein thelightguide comprises a first terminus proximate to the detector and asecond terminus distal to the detector;

(f) a second emission lightguide for transmitting emission from thefluorescent species to the second emission detector, wherein thelightguide comprises a first terminus proximate to the detector and asecond terminus distal to the detector;

(g) a probe housing the distal termini of the excitation lightguide,first emission lightguide, and second emission lightguide; and

(h) a housing for receiving the probe, wherein the housing is adaptedfor receiving the probe at a first end and terminating with a window atthe second end, the window being transparent to the excitation and theemission light, and wherein the window physically isolates the probefrom the fluorescent species.

In one embodiment, the system further includes a means for writing pHdata to the vessel containing the sample, the vessel having a means forreceiving the pH data that further allows the data to be read from thevessel at a later time.

In one embodiment, the first and second detectors are the same. In oneembodiment, the first and second detectors are different. In oneembodiment, the first and second detectors are photodiodes.

In one embodiment, the first emission lightguide and the second emissionlightguide are the same. In one embodiment, the first emissionlightguide and the second emission lightguide are different. In oneembodiment, the excitation lightguide, the first emission lightguide,and the second emission lightguide are optical fibers.

There are five basic optical chemical sensing techniques: measuringabsorbance, fluorescence intensity, ratiometric fluorescence,fluorescence lifetime, and fluorescence polarization. Sample parameters,such as pH, can be measured using any of the five techniques. Thesystems and methods described herein utilize fluorescence sensing andcan be used to monitor sample parameters that produce changes influorescence intensity, ratiometric fluorescence, fluorescence lifetime,and fluorescence polarization characteristics of a fluorescent speciesin liquid contact with the sample to be monitored.

In one embodiment, the method is a fluorescent wavelength ratiometricmethod. As used herein, the term “fluorescent wavelength-ratiometric”refers to a method by which the first and second fluorescent emissionintensities of a fluorescent species are measured at first and secondemission wavelengths, respectively, and are ratioed to provide pHinformation. In this embodiment, the fluorescent species has emissionintensities that vary as a function of pH.

In one embodiment, the fluorescent species is a ratiometric fluorescentspecies. In one embodiment, the fluorescent species is selected from anaphthofluorescein compound and a seminaphthorhodamine compound. In oneembodiment, the naphthofluorescein compound is selected from aseminaphthofluorescein compound and a carboxynaphthofluoresceincompound. In one embodiment, the seminaphthofluorescein compound isselected from 5′(and6′)-carboxy-3,10-dihydroxy-spiro[7H-benzo[c]xanthene-7,1′(3′H)-isobenzofuran]-3′-one(also referred to herein as “SNAFL-1”, see FIG. 23A) and2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoicacid (also referred to herein as “EBIO-3”, see FIG. 23E).

In one embodiment, the fluorescent species is immobilized on a substrateand comprises a conjugate of a fluorescent species and a macromolecule.In one embodiment, the macromolecule is an albumin. In one embodiment,the macromolecule is a serum albumin. In one embodiment, themacromolecule is a human serum albumin. In one embodiment, themacromolecule is a recombinant human serum albumin. In one embodiment,the fluorescent species immobilized on a substrate comprises anaphthofluorescein/serum albumin conjugate. In one embodiment, thefluorescent species immobilized on a substrate comprises aseminaphthofluorescein/human serum albumin conjugate.

As noted above, the method is suitable for measuring the pH of blood orblood products contained in a sealed vessel. In one embodiment, thefluorescent species (e.g., substrate-immobilized fluorescent species) isintroduced into a sealed vessel during the vessel's manufacture andbefore the sample is introduced to the vessel. In one embodiment, thefluorescent species is introduced into a sealed vessel by a means thatpreserves the vessel's seal.

The method and system are useful for monitoring a parameter of a sample,for example, the pH or CO₂ of blood or blood products. The method andsystem are suited to monitor the pH of a sample contained in a sealedvessel.

The presence of microorganisms growing in PCs causes dynamic changes invarious parameters in the mixture, such as pH, CO₂, O₂, and glucoseconcentrations. These parameters can be analyzed as surrogate markersfor the presence of microbiological contamination. A non-invasive“in-the-bag” sensor, used to measure chemical parameters such as pH, CO₂levels, O₂ levels, or glucose concentrations provides greatersensitivity for detecting perturbations in the bag environment caused bythe growth of microorganisms due to the elimination of samplingartifacts and the ability to carry out multiple measurements over timewithin a closed system. The systems and methods can detect subtlechanges in pH or CO₂ within the closed system or the bag environment,and can be monitored continuously or periodically over time withoutdirect sampling.

In one embodiment, a method of monitoring pH of a sealed sterileplatelet storage device over time and analyzing the changes to predictthe quality of the platelet rich plasma and/or platelet rich plasmaadditive solution formulations is provided. The non-invasive methodsdescribed herein enable this “real-time” analysis of platelet quality.Previously, samples had to be physically withdrawn from the plateletstorage bag and analyzed on a blood gas analyzer or a separateinstrument. After puncturing the bag, the PC must be used within 4 hoursdue to risk of contamination. Preserving the unit required laborioussampling of bag contents via a long plastic tube attached to the bag(pigtail) using a sterile sealer device to remove portions thereof.

The data presented herein was obtained using a small platelet storagebag with a 14 mL storage volume having a built-in pH reading insert. Alarger storage bag for a transfusible unit is designed to hold 300 mL ofpooled platelet concentrate. This larger bag could be used for plateletsrecovered from a single donor by apheresis or for storage of apre-storage pooled platelet concentrate unit.

Large and small platelet storage bags with built-in inserts are shown inFIGS. 1 and 2, respectively. Referring to FIG. 1, large storage bag 500includes a plurality of vessel ports 510 and port assembly 232. Portassembly 232 is further described in FIG. 22. Storage bag 500 alsoincludes label 1300, into which a memory device can be integrated (notshown). By integrating a memory device into label 1300, parameterreadings can be written to and stored on storage bag 500 that containsthe sample.

Referring to FIG. 2, small storage bag 500 includes a plurality ofvessel ports 510 and port assembly 232. Port assembly 232 is furtherdescribed in FIG. 22. Storage bag 500 also includes label 1300, intowhich a memory device can be integrated (not shown). By integrating amemory device into label 1300, parameter readings can be written to andstored on storage bag 500 that contains the sample.

The pH reading accuracy of the non-invasive platelet bag inserts andmeasuring system described herein have been tested. FIGS. 3A-3D aregraphs showing pH profiles of four individual PC samples. The PCs werestored in representative large sterile storage bags containing 300 mL ofpooled platelet concentrates per bag. These platelets were leukoreducedprior to storage in the bags, and the four bags tested all showed goodplatelet health parameters over the 11 days of storage. The bags werespiked in an aseptic manner with sample site adaptors (Codan) via thetwist off ports incorporated on the bags to allow samples to bewithdrawn for analysis on a blood gas analyzer.

Referring to FIGS. 3A-3D, two pH profiles are shown for each sample. ThepH profiles for each sample were generated from two pH measurementsystems, namely, a non-invasive fluorescent pH reading and an invasiveblood gas pH reading. In each figure, the pH profile generated fromfluorescent pH readings is designated “BCSI pH” and shown as a solidline. The pH profile generated from blood gas pH readings is designated“Blood Gas pH” and shown as a dashed line. The data show similar pHprofiles for these “normal” platelet concentrates over 11 days ofstorage. This demonstrates that the bags with inserts can be ethyleneoxide sterilized without compromising pH reading performance. This alsodemonstrates that the accuracy of the two pH measurement systems(non-invasive fluorescent reading vs. invasive blood gas reading) wasconsistent. The mean difference from average pH determined by bothmethods was 0.045 (n=32, SD 0.058). This is the first demonstration of asystem for measuring the metabolic activity in the contents of aplatelet storage bag in a non-invasive manner over time.

To determine the suitability of the representative manufactured bags forstorage of platelet concentrates, the four sterile representative bagsdescribed above were compared with similar sized control sterileplatelet storage bags from Fresenius (The Netherlands). Two 300 mLpooled platelet rich plasma units were leukoreduced, combined anddistributed to the paired storage bags. The bags were spiked in anaseptic manner with sample site adaptors (Codan) via the twist off portsincorporated on the bags to allow samples to be withdrawn for analysison a blood gas analyzer. The results, shown in FIGS. 4-7, demonstratevirtually identical platelet storage parameters for the pairedrepresentative (“BCSI”) and control (“Fresenius”) bags.

Referring to FIGS. 4A and 4B, platelet shape parameters for PCs storedin representative and control platelet storage bags were evaluated. FIG.4A is a graph comparing Kunicki Scores of PCs stored in representativeand control platelet storage bags for 9 days. The Kunicki Scores in FIG.4A range in value from 0 to 400. A Kunicki Score of 0 indicatesballoon-shaped, or dead platelets; 100 indicates dendrites, or anactivated platelets; 200 indicates spheres, or spherical platelets; and400 indicates discoid platelets. The results show virtually identicalstorage parameters for the paired representative and control bags.

FIG. 4B is a graph comparing Swirling Scores for platelets stored inrepresentative and control platelet storage bags for 9 days. TheSwirling Scores in FIG. 4B range in value from 0 to 3. A Swirling Scoreof 0 indicates dead platelets; a score of 2 is the standard value forplatelets at 6 days of storage; and a score of 3 indicates very goodplatelets. The results show virtually identical storage parameters forthe paired representative and control bags.

Referring to FIGS. 5A and 5B, the pH, glucose, and lactate profiles forPCs stored in representative and control platelet storage bags weredetermined. FIG. 5A is a graph comparing pH profiles of platelets storedin representative and control platelet storage bags for 9 days. Theresults show virtually identical storage parameters for the pairedrepresentative and control bags.

FIG. 5B is a graph comparing glucose and lactate concentration profilesof platelets stored in representative and control platelet storage bagsfor 9 days. The glucose concentrations are depicted with diamond-shapedpoints and range from approximately 20 mM at storage day 1 toapproximately 10 mM at day 9. The lactate concentrations are depictedwith triangle-shaped points and range from approximately 6 mM at storageday 1 to approximately 17 mM at day 9. The results show virtuallyidentical storage parameters for the paired representative and controlbags.

Referring to FIGS. 6A and 6B, the metabolic function of platelets storedin representative and control bags was determined. FIG. 6A is a graphcomparing ATP:ADP ratio profiles for platelets stored in representativeand control platelet storage bags for 9 days. The ATP:ADP ratio ismeasured on a scale of 1 to 10. An ATP:ADP ratio of 1 indicates aresting sample, and a ratio of 10 indicates a metabolically activesample. The results show a typical ATP:ADP response for the pairedrepresentative and control bags.

FIG. 6B is a graph comparing mitochondrial potential profiles forplatelets stored in representative and control platelet storage bags for9 days. The mitochondrial potential is measured on a scale of 0 to 4. Amitochondrial potential reading of 0 indicates damaged platelets, and amitochondrial potential reading of 3 indicates good platelets. Theresults show good mitochondrial potential throughout the test period forthe paired representative and control bags.

Referring to FIG. 7, platelet activation levels were measured for PCs inrepresentative and control platelet storage bags for 9 days. FIG. 7 is agraph comparing the level of platelet activation measured by CD62P andAnnexin V percentages. The CD62P percentages are depicted with thediamond-shaped points and range from approximately 5% at storage day 2to approximately 17% at day 9. The Annexin V percentages are depictedwith triangle-shaped points and range from approximately 5% at storageday 4 to approximately 16% at day 9. The results are consistent withmoderate platelet activation in paired representative and control bags.

The pH over time for platelets stored in representative small plateletstorage bags was examined. Referring to FIG. 8, pH profiles weregenerated for normal and under-filled small platelet storage bags. Thenormal bags contained 14 mL of apheresis platelets. The under-filledbags contained 7 mL of apheresis platelets from the same unit as thenormal bags. The best storage conditions used 14 mL of PC per bag, andreproducible curves were obtained for these “normal” storage conditions.As shown in FIG. 8, if the bags are under-filled (7 mL of PC per bag),then the pH profile changes such that the pH drops more rapidly. This isthe first demonstration that the non-invasive system for measuring themetabolic activity in the contents of a platelet storage bag over timecan be used to find “abnormal” units. Other storage parameters such asabnormal leukocyte count or abnormal platelet concentration or novelparameters that influence platelet storage may also be detected usingthe non-invasive method. It is recognized that platelets recovered fromsome donors rapidly lose function and it is likely that these units canbe discovered using a non-invasive system.

Bacterial contamination of platelets is a common problem, and the mostlife threatening of all blood transfusion events. Results ofnon-invasive pH measurements of small platelet bags inoculated with fourcommon bacterial contaminants are shown in FIGS. 9A-9D. The bags wereinoculated at 26 hours and detected at the inflection time point shownin each graph. All bags were incubated at 22° C. FIG. 9A represents a PCsample inoculated with 10 CFU/mL (Colony Forming Units/mL) of E. coli.The sample at 72 hours contained 3.7E4 CFU/mL bacteria. FIG. 9Brepresents a PC sample inoculated with 10 CFU/mL of K. oxytoca. Thesample at 48 hours contained 3.3E5 CFU/mL of bacteria. FIG. 9Crepresents a PC sample inoculated with 90 CFU/mL S. marcescens. Thesample at 48 hours contained 3.6E3 CFU/mL of bacteria. FIG. 9Drepresents a PC sample inoculated with 240 CFU/mL S. aureus. The sampleat 60 hours contained 6.2E7 CFU/mL of bacteria.

Referring to FIGS. 9A-9D, pH was measured using a Bayer blood gasanalyzer and a system and method described herein. All organisms testedin this cohort show a dramatic decrease in pH with ongoing bacterialgrowth. Concomitant invasive sampling with the blood gas analyzerconfirmed the accuracy of the non-invasive pH measurement system. The“detection point” shown in FIGS. 9A-9D can be correlated with bacterialcount upon examination of the rate of change in pH per unit of time overthe course of the bag tracking pH measurements. The “detection point”can also be correlated with other chemical parameters or numericalratios thereof as measured using various methods in these experiments.More frequent sampling would likely detect the bacteria at lower CFU.

The value of measuring other analytes associated with metabolizingplatelets in storage bags is shown in FIGS. 10A-10D. Small storage bagswere inoculated with 10 CFU/mL of K. pneumoniae. Partial pressures ofoxygen and carbon dioxide were measured on a blood gas analyzer atvarious time points. Glucose concentrations were measured using acommercial dipstick method at the same time points. The pH measurementsin FIG. 10A show paired blood gas and pH readings for a singleinoculated bag. The O₂, CO₂, and glucose graphs in FIGS. 10C, 10D, and10B, respectively, compare inoculated and “normal” plateletconcentrates. Bacterial contamination of the platelet concentrates canalso be correlated with these other chemical parameters, or numericalratios thereof, as measured using various methods in these experimentsas shown in FIGS. 10A-10D.

FIG. 11 illustrates a representative multi-probe platelet storage bagwith two inserts, one for measuring pH and the other for measuring CO₂.Referring to FIG. 11, storage bag 500 includes a plurality of vesselports 510, port assembly 232, and port assembly 232. Storage bag 500includes label 1300, into which a memory device can be integrated (notshown). By integrating a memory device into label 1300, parameterreadings can be written to and stored on storage bag 500 that containsthe sample.

Thus, in one embodiment, a method of monitoring (e.g., measuring andrecording) at least two chemical parameters in a sealed sterile plateletstorage device is provided. In the method, the parameters monitoredindicate platelet healthiness (e.g., low pH) or microbial contamination(e.g., CO₂ spike).

Storage of the platelet units at 22° C. in a shaking incubator isstandard in all blood banks worldwide. Although fluorescence readers canbe used to rapidly measure pH, it still requires individual handling ofevery bag at each time point. To alleviate this problem, in oneembodiment, an incubator that facilitates automated reading is provided.

In one embodiment, a single reader incubator with a flexible probe isprovided. The probe is mechanically moved to different positions on theback of the incubator using an x,y,z robot. Referring to FIG. 12, anoptical platform 180 is located proximate to a shelving unit that isconfigured to receive platelet storage bags 500. The optical platform180 is more fully described below (see FIG. 19). Platelet storage bags500 are placed on shelves 5100 by a user. A plurality of apertures 5200are disposed towards the distal end of each shelf. Clamps 5300 arelocated at each aperture 5200. Each bag 500 may be secured through anaperture 5200 using clamp 5300. A probe 185 is connected by opticalfibers (lightguides) to optical platform 180. Probe 185 is enabled tomaneuver within an x,y,z grid. The z dimension involves reversibleinsertion of the probe 185 into the clamped insert for each bag 500 at aspecified x,y position. Any number of bags may be placed in theincubator provided there are sufficient apertures 5200 to receive them.

In another embodiment, a single reader incubator is configured withmultiple fiber bundles. Referring to FIG. 13, multiple bags 500 withsensors are each connected to separate probes 185 by a user and leftattached to the probes. Separate probes 185 are connected to opticalplatform 180 by optical fibers (lightguides). Optical platform 180 ismore fully described below (see FIG. 19). The fiber optics in the probes185 convey excitation light to the sensor and fluorescent emissions backto the detector within the optical platform 180. The excitation lightcan be split from one light source and piped to multiple probes 185 andbag sensors by physically blocking the excitation fiber optics so thefiber optic for only one probe is excited.

For example, if four probes (1, 2, 3, and 4) are connected to a singleexcitation source a moving shutter blocks the light collection ends ofprobes 1, 2 and 3 to only allow light to travel to probe 4. Then theshutters are changed to block the ends of probes 2, 3, and 4 allowingexcitation only to probe 1. Each of the dual emissions from the sensorsare collected with light for each channel from all the probes focused ona single detector. For example, a system with four probes (1, 2, 3, and4) and emission readings A and B for each probe, would have twodetectors A and B. Detector A would have all the light from probe 1A,2A, 3A, and 4A focused on it and likewise with detector B. Thendepending on what probe was excited by means of the physical blocking,the emission readings can be tied to which probe was excited. Aplurality of bags could be studied with a single excitation and emissionsystem in this way also a plurality of these excitation and emissionsystems 700 could be incorporated into a single central processor 170,with output to user interface and display 130.

FIG. 14 is a schematic illustration for the incubators described inFIGS. 12 and 13. Referring to FIG. 14, the schematic illustration showshow the components of a representative system for carrying out themethod are adapted and connected to make an incubator. System componentsinclude display 130 for determining the status of the system and viewingpH determination results; memory device 165 for storing test results andcalibration data; signal processing electronics 170 for commanding theoptical platform components and processing signals from the opticalplatform; and optical platform 180 including an excitation source 280,emission detectors 380 and 480, lightguides, and associated lenses andfilters. Optical platform 180 includes probe 185. Probe 185 isreversibly connectible to bag 500 with insert.

In one embodiment, an incubator machine may have one probe 185 thatconnects to multiple bags 500, as shown in FIG. 12. In anotherembodiment, an incubator machine may have multiple probes 185 and asingle optical platform 180, as shown in FIG. 13. In yet anotherembodiment, an incubator machine may have multiple probes 185 withmultiple excitation sources 280 and emission detectors 380 and 480. Thebasic structure for this embodiment is shown in FIG. 14.

To have practical utility, the parametric information (e.g., pH)obtained by the systems and methods need be stored and analyzed. Thereare several options for storing and analyzing the obtained data.Multi-read data can be stored for real-time analysis on a memory device,such as a chip, that is integrated into the storage bag (i.e., vessel)itself. The memory device is read/write capable in order to receiveinformation and store it for later analysis. In this way, data aredirectly linked to the bag and follow the bag from one location to thenext. For example, a bag may be transferred from a donor center to aresearch laboratory or hospital blood bank. Storing the data on a memorydevice that is integrated into the bag allows the data to be readdirectly from the storage bag prior to being analyzed.

In one embodiment, data of the pH and other analytes can be stored on amemory device attached to the platelet bag. A Texas Instruments S2000RFID transponder and corresponding low-frequency RFID tags were employedas a proof of principle platform. The available RFID tags were limitedin memory capacity, having at most 80 bits available for read/writeoperations. To overcome this memory limitation, pH values were encodedto have a range such that pH 6.2 and 7.8 corresponded to hexadecimalvalues zero (0) and fifteen (F), respectively. In binary representationeach hexadecimal value requires four bits.

A representative system (BCSI pH1000, Blood Cell Storage, Inc., Seattle,Wash.) was coupled to the RFID system through a personal computer. AnRFID antenna was fitted inside the pH1000 casing. Software was writtenin Matlab to integrate the functions of the pH1000 and S2000. When atransponder entered the antenna range, data was automatically read fromthe RFID tag and the user prompted to perform a reading. Subsequent toadditional measurements on the pH1000; new data was appended to the RFIDtag.

Bags can be identified and tracked by integrating an RFID chip in thebag itself as a means for writing data to the vessel containing thesample. Referring to FIGS. 1 and 2, the chip can be integrated in thelabel 130 of bag 500. Bags can also be identified and tracked byidentifying each bag with a unique bar code. The bar code on theplatelet storage bag can also be used for other identificationinformation, for example, to identify the bag's position in theincubator. When data are stored on an RFID chip, there is no need toidentify the bag position in the incubator.

Another data storage option is to store data on a central computer thatis linked to the system for monitoring pH. When data are collected, theyare transferred to a central computer where they are stored. The centralcomputer is equipped to generate a pH profile using data that it hasaccumulated. Yet another option is to store data directly on the reader.Data can be processed on the reader to generate a pH profile, or can betransferred to a central computer that generates the pH profile.

To advise regarding the quality of the sample in the vessel interrogatedby the systems and methods, pass/fail algorithms may be utilized thatexamine the rate of change in pH per unit of time over the course of thebag tracking in an automated fashion. A drop in pH greater than somethreshold value as defined from examination of a plurality of data wouldconstitute assignment and reporting of a FAIL value which will beassociated with the particular bag being examined.

In one aspect, a method for monitoring pH of a sample is provided. Inthe method, pH is determined by comparing fluorescent emissionintensities from a single fluorescent species having pH-dependentfluorescent emission. The fluorescent species having pH-dependentfluorescent emission has a first emission intensity at a firstwavelength and a second emission intensity at a second wavelength, thefirst and second emission intensities being characteristic of pH in theenvironment of the fluorescent species. The ratio of the first andsecond emission intensities provides pH measurement. Calibration of thefirst and second emission intensities provides an intensity-basedreference (ratio information) that is used to determine the pH of theenvironment of the fluorescent species.

In one embodiment, fluorescent signal analysis of the ratio of first andsecond emission wavelengths is used to calculate the resulting pH.Fluorescent signal analysis and analysis of the pH algorithm areperformed as follows. The instrument collects the fluorescentintensities at 568 nm (fr) and at 600 nm (fp), and then calculates theratio of the fp to fr signals. The pH has been found to be a function ofthe ratio and the fp value described as pH=f(ratio)+g(fp). The f(ratio)function is nonlinear while the g(fp) function is linear. The bestdescription of the f(ratio) is Constant1*ln(ratio)+Constant2. Theinstrument then uses a lookup table, such as the lookup table in FIG.15, to find the appropriate value for the f(ratio) function and thencalculates the g(fp) adjustment and calculates a pH. Because theintensities of the fr and fp channels are dependent on optical path andthe instrument hardware, the intensity values obtained from a singlepart are not identical for each instrument. However, the differencesbetween these intensities are linearly related and thus it is possibleto compare the results from one instrument to another as well as createthe f(ratio) and g(fp) relationships on a different instrument than theinstrument that the calibration of pH and fluorescent signal wasperformed on.

FIG. 15 is a graph of a mathematical function of the ratio of first andsecond emission intensities as related to pH. This type of graph is alsoknown as a lookup table, and is useful in the method and systemdescribed herein. For example, according to the lookup table in FIG. 15,a ratio of 1.6 corresponds to pH 6.0; a ratio of 2.56 corresponds to pH7.0; and a ratio of 4.48 corresponds to pH 8.0.

The lookup table in FIG. 15 is generated from calibration data depictedin FIG. 16. FIG. 16 shows the data from calibration studies of fivelots. Individual lookup tables are possible for each lot or as FIG. 16demonstrates the lots when compared to one lookup table agree within±0.159 pH units. Calibration data is collected in the manner describedin Example 7. Apheresis platelets and plasma are separated into largeplatelet bags rigged to resemble the small bag (see FIG. 2). Four groupsare followed over time to give a suitable range of pH. The four groupsare: (1) normal—standard fill of 12-15 mLs to generate pH decline overthe shelf life of the platelets, (2) plasma—to generate high pH range,(3) underfills—fill of only 7 mLs, and (4) bacterially spiked—standardfill with the addition of 100 CFU/mL of Klebsiella oxytoca after the 24hour read to generate very low pH. Readings are initially performedafter 4 hours of equilibration, then twice daily through four days andonce daily for the remainder of 7-10 days.

Platelet pH trends were analyzed to determine acceptable andunacceptable rates of change for stored platelets. The rate of changecan be used to determine the quality of stored PC. Referring to FIG. 17,pH profiles were generated for 5 types of platelets: PC sample with alow platelet count, PC sample with a high platelet count, two PC sampleswith bacterial contamination, and PC sample that is a “poor storer.”After an initial rise in pH attributable to CO₂ off gassing, pH rates ofchange can be described as follows. PC samples with a high plateletcount had a pH drop of about 0.02 pH units per day. PC samples with alow platelet count had a pH drop of about 0.04 pH units per day. pHdrops in this range are characteristic of good quality PCs that areusable. In comparison, poor storing PCs had a pH drop of about 0.08 pHunits per day, and bacterially-contaminated PCs had a pH drop of about0.2 pH units per day. pH drops in this range are characteristic of poorquality PCs that are unusable.

To further understand the systems and methods, the following detaileddescription is provided.

In one embodiment, the fluorescent species having pH-dependentfluorescent emission is immobilized on a substrate in contact with thesample such that the fluorescent species is in contact with the sample.The immobilized fluorescent species in contact with the sample islocated in the sample such that the fluorescent species can beinterrogated. The fluorescence measurement is made by irradiating thefluorescent species at a wavelength sufficient to elicit fluorescentemission, which is then measured. Because of the pH-dependent nature ofthe fluorescent species' emission profile (i.e., first and secondfluorescent emission intensities measured at first and second emissionwavelengths, respectively) the measurement of the fluorescent emissionprofile yields the pH of the fluorescent species' environment (i.e.,sample pH).

In one embodiment, the sample for which the pH is to be determined iscontained in a sealed vessel. As noted above, this method is suitablefor measuring pH of blood and blood products sealed in a conventionalblood storage vessel.

In another embodiment, the sample for which the pH is to be determinedis contained in an open vessel. As used herein, the term “open vessel”refers to a vessel that is not sealed. In this method, the probe iscleaned and/or sterilized and is used once and discarded. This method issuitable for measuring the pH of materials used in food, pharmaceutical,or biological research where the vessel containing the material is notsealed (i.e., open). Such a “lab-use” system includes a tip (seedescription below) placed onto the probe. The pH measurement is made byimmersing the tip into the sample and measuring pH. The tip is removedfrom the sample, removed from the probe, and discarded.

In the method for measuring the pH of a contained sample, thefluorescent species (e.g., substrate-immobilized fluorescent species) isintroduced into the vessel either before or after the sample is placedin the vessel. The sealed vessel prevents the contents of the vesselfrom contact from, for example, liquids, gases, or contaminants outsideof the vessel. The sealed vessel also prevents the contents of thevessel from escaping the vessel.

The vessel can be manufactured to include the substrate-immobilizedfluorescent species as a component of the vessel. In such an embodiment,the substrate-immobilized fluorescent species is incorporated into thevessel during manufacture to provide a vessel into which a sample can belater introduced and its pH measured. The manufacture of a vesselincorporating the substrate-immobilized fluorescent species is describedin Example 1.

Alternatively, the substrate-immobilized fluorescent species can beintroduced into the vessel after the sample has been introduced into thevessel. In such an embodiment, the substrate-immobilized fluorescentspecies is introduced into the vessel by a process in which the vesselis first punctured (or spiked) to introduce the substrate-immobilizedfluorescent species and then resealed to provide a sealed vesselincluding the sample now in contact with the substrate-immobilizedfluorescent species. The process for introducing thesubstrate-immobilized fluorescent species into a sealed vessel isdescribed in Example 2.

As noted above, the vessel including the substrate-immobilizedfluorescent species in contact with the sample is sealed before, during,and after interrogation. Interrogation of the fluorescent speciesrequires excitation of the species at a wavelength sufficient to effectfluorescent emission from the species and measurement of thatfluorescent emission. In the method, interrogation is accomplishedthrough a window in the sealed vessel. The fluorescent species isexcited by irradiation through the window, and emission from thefluorescent species is collected from the fluorescent species though thewindow. The window is a component of the sealed vessel and allows forinterrogation of the fluorescent species in contact with the sample. Thewindow is sufficiently transparent at the excitation and emissionwavelengths to permit interrogation by the method. Thesubstrate-immobilized fluorescent species is positioned in proximity tothe window sufficient for interrogation: proximity sufficient toeffectively excite the fluorescent species and to effectively collectemission from the fluorescent species. It will be appreciated that forepifluorescence applications, a single window is used. However, othermethods and devices can include other optical paths, such asstraight-through or right angle optical paths, where more than onewindow can be used.

The method includes irradiating the fluorescent species, which in oneembodiment is contained along with a sample in a sealed vessel, at awavelength sufficient to effect emission from the fluorescent speciesand to measure that emission. Exciting light and fluorescent emissionpass through the sealed vessel's window. In one embodiment, the sealedvessel further includes a port for receiving a housing that holds theexcitation lightguide and emission lightguide. In one embodiment, theexcitation lightguide includes one or more optical fibers that transmitthe excitation light from a light source to the fluorescent species. Inone embodiment, the emission lightguide includes one or more opticalfibers that transmit the emission light from the fluorescent species toa light detector. The port receiving the housing is positioned inproximity to the window sufficient for interrogation: proximitysufficient to effectively excite the fluorescent species and toeffectively collect emission from the fluorescent species.

As with all optical fluorescent methods, the method includes a lightsource for exciting the fluorescent species and a detector for measuringthe emission of the fluorescent species. Light sources, wavelengthselection filters, and detectors are selected based on the absorbanceand emission profiles of the fluorescent species used in the method.

Suitable light sources provide excitation energy at a wavelength andintensity sufficient to effect fluorescent emission from the fluorescentspecies. The light source can provide relatively broad wavelength bandexcitation (e.g., ultraviolet or white light sources) or relativelynarrower wavelength band excitation (e.g., laser or light-emittingdiode). To enhance excitation efficiency and emission measurement,relatively broad wavelength band exciting light from the source can beselected and narrowed through the use of diffraction gratings,monochromators, or filters to suit the fluorescent species. Suitablelight sources include tungsten lamps, halogen lamps, xenon lamps, arclamps, LEDs, hollow cathode lamps, and lasers.

Suitable detectors detect the intensity of fluorescent emission over theemission wavelength band of the fluorescent species. To enhance emissionmeasurement, fluorescent emission from the fluorescent species sourcecan be selected and narrowed through the use of diffraction gratings,monochromators, or filters to suit the fluorescent species. Suitabledetectors include photomultiplier tubes and solid state detectors, suchas photodiodes, responsive to the wavelength emission band of thefluorescent species. Other suitable detectors are photovoltaic cells,PIN diodes, and avalanche photodiodes.

Through the use of filters, all of the excitation light that reflectsoff the target is filtered out before reaching the detector. This can beachieved by using filters in both the excitation and emission opticalpaths. In certain instances, reflected excitation light (which is manyorders of magnitude more intense than the emission light) that reachesthe detector can swamp the specific signal. Generally, 10E5 (10⁵) orgreater out-of-band rejection is appropriate in each of the filter sets.Reduction of excitation light can also be achieved by using an angledwindow so that reflected light is directed away from the emissiondetector. However, such an optical path is not as effective as filtersets.

Excitation light from the source can be directed to the fluorescentspecies through the use of a lightguide, such as one or more opticalfibers. Similarly, emission from the fluorescent species can be directedto the detector through the use of a lightguide, such as one or moreoptical fibers.

A representative system for carrying out the method is illustratedschematically in FIG. 18. Referring to FIG. 18, system 100 includescontroller 110 that controls and operates the system components. Systemcomponents include keypad 120 for inputting information including systemcommands; display 130 for determining the status of the system andviewing pH determination results; barcode reader 140 for inputtinginformation to the system including the identification of the sample,the pH of which is to be measured by the system; printer 150 forprinting system status and pH determination results; battery (or wallplug and power adapter) 160 for powering the system; memory device 165for storing test results and calibration data; signal processingelectronics 170 for commanding the optical platform components andprocessing signals from the optical platform; and optical platform 180including an excitation source, emission detectors, lightguides, andassociated lenses and filters. Optical platform includes probe 185housing one or more excitation lightguides and two or more emissionlightguides. FIG. 19A also illustrates sealed vessel 500 including port205 for receiving probe 185.

FIG. 19 is a schematic illustration of an optical platform useful in thesystem for measuring pH. Referring to FIG. 19, optical platform 180includes excitation optics 280, first emission optics 380, and secondemission optics 480. Excitation optics 280 include light source 282,collimating lens 284, filter 286, focusing lens 288, and excitationlight waveguide 290. First emission optics 380 include detector 382,focusing lens 384, filter 386, collimating lens 388, and first emissionlight waveguide 390. Second emission optics 480 includes detector 482,focusing lens 484, filter 486, collimating lens 488, and second emissionlight waveguide 490. Excitation lightguide 290, first emission lightwaveguide 390, and second emission light waveguide 490 are housed inprobe 185.

The system's light source is effective in exciting the fluorescentspecies. Suitable light sources include light-emitting diodes, lasers,tungsten lamps, halogen lamps, xenon lamps, arc lamps, and hollowcathode lamps. In one embodiment, the light source is a light-emittingdiode emitting light in the range from 500 to 560 nm. A representativelight-emitting diode useful in the system is a green ultrabright Cotco503 series LED commercially available from Marktech, Latham N.Y.

The collimating lens directs light (e.g., excitation light from thelight source or first and second emission light from the emission lightwaveguides) to the bandpass filter. Suitable collimating lenses includeBiconvex glass lenses and Plano-convex glass lenses. Representativecollimating lenses useful in the system are the Tech Spec PCX lensescommercially available from Edmund Optics, Barrington, N.J. Theexcitation collimating lens is 12×36 (diameter by effective focal lengthin mm) and the first and second emission collimating lenses are 12×18.

The focusing lens focuses light from the bandpass filter to theexcitation light waveguide or from the bandpass filter to the detector.Suitable focusing lenses include Biconvex glass lenses and Plano-convexglass lenses. Representative focusing lenses useful in the system arethe Tech Spec PCX lenses commercially available from Edmund Optics,Barrington, N.J. The excitation focusing lens is 12×18 and the first andsecond emission focusing lenses are 12×15.

Filters are used in the optical platform to narrow the bandwidth oftransmitted light.

Suitable excitation filters include bandpass filters, shortpass filters,longpass filters, or a combination of short and long pass filters. Inone embodiment, the system uses a shortpass filter that passes light inthe range from about 370 nm to 540 nm. A representative excitationshortpass filter useful in the system is 540ASP commercially availablefrom Omega Optical, Brattleboro, Vt.

Suitable first emission filters include bandpass, shortpass, longpass,or a combination of short and longpass filters. In one embodiment, thebandpass filter passes light in the range from about 595 to 605 nm andhas a full width at half height of 10 nm. A representative firstemission bandpass filter useful in the system is 600DF10 commerciallyavailable from Omega Optical, Brattleboro, Vt.

Suitable second emission filters include bandpass, shortpass, longpass,or a combination of short and longpass filters. In one embodiment, thebandpass filter passes light in the range from about 562 to 573 nm andhas a full width at half height of 10 nm. A representative secondemission bandpass filter useful in the system is 568DF10 commerciallyavailable from Omega Optical, Brattleboro, Vt.

The excitation light waveguide transmits excitation light from the lightsource through the probe to the fluorescent species. In one embodiment,the excitation light waveguide includes one or more optical fibers. Inone embodiment, the excitation waveguide is a single optical fiber. Arepresentative fiber optic useful in the system is RO2-534 commerciallyavailable from Edmund Optics, Barrington, N.J.

The first and second emission light waveguides transmit fluorescentemission from the fluorescent species through the probe to the first andsecond emission detectors, respectively.

In one embodiment, the first emission light waveguide includes one ormore optical fibers. In one embodiment, the first emission lightwaveguide includes a plurality of optical fibers. In one embodiment, thefirst emission light waveguide includes four optical fibers. Arepresentative fiber optic useful in the system is RO2-533 commerciallyavailable from Edmund Optics, Barrington, N.J.

In one embodiment, the second emission light waveguide includes one ormore optical fibers. In one embodiment, the second emission lightwaveguide includes a plurality of optical fibers. In one embodiment, thesecond emission light waveguide includes four optical fibers. Arepresentative fiber optic useful in the system is RO2-533 commerciallyavailable from Edmund Optics, Barrington, N.J.

Suitable optical fibers useful in the system include glass or plasticoptical fibers from 0.2 to 2 mm diameter.

The system's first and second emission detectors are effective inmeasuring the first and second fluorescent emissions from thefluorescent species. Suitable detectors include photodiodes, PIN diodes,and photomultiplier tubes. In one embodiment, the first and secondemission detectors are photodiodes responsive in the range from 400 to800 nm. Representative photodiodes useful in the system include BPW34commercially available from Vishay Intertechnology, Malvern, Pa.

A representative probe housing excitation and emission lightguidesuseful in the system is illustrated schematically in FIG. 20. As shownin FIG. 20, the lightguides are optical fibers. Referring to FIG. 20,probe 185 houses excitation lightguide 290, a plurality of firstemission lightguides 390, and a plurality of second emission lightguides490. In the representative probe shown in FIG. 20, there are four firstemission lightguides 390, and four second emission lightguides 490. Thefour first emission lightguides can be considered to be a first channel(e.g., measuring the first fluorescent emission from the fluorescentspecies) and the four second emission lightguides can be considered tobe a second channel (e.g., measuring the second fluorescent emissionfrom the fluorescent species). In the illustrated representative probe,the fibers from each of the two sets of fibers alternate (i.e.,alternating fibers 390 and 490) around the central fiber (290). Thisconfiguration provides for evening out of “hot spots” so that lightcollected by the first set is similar to the light collected by thesecond set.

The relationship between the probe housing the excitation/emissionlightguides and the sealed vessel port is illustrated schematically inFIG. 21. Referring to FIG. 21, probe 185 is received by port 205. Port205 includes window 210, which is transparent to excitation and emissionwavelengths used in the fluorescent measurement. Excitation lightemanating from lightguide 290 passes through window 210 and interrogatessubstrate 220 on which the fluorescent species is immobilized and which,in the operation of the method, is in contact with the sample containedin sealed vessel 200. Irradiation of substrate 220 results in excitationof the substrate-immobilized fluorescent species and fluorescentemission from the fluorescent species. Emission from the fluorescentspecies is received by and transmitted through lightguides 390 and 490to detectors 382 and 482, respectively (see FIG. 19B). As noted above,the fluorescent species' first emission intensity and the secondemission intensity will depend on the pH of the sample.

A representative port assembly useful for incorporation into a sealedvessel during manufacture is illustrated in FIG. 22. Referring to FIG.22, port assembly 232 includes port 205 and tip 235. Port 205 is acylinder terminating with window 210 and having opening 212 forreceiving probe 185 (not shown). In one embodiment, port 205 tapers fromopening 212 to window 210 such that the depth of insertion of probe 185into port 205 is predetermined by the probe's diameter. When inserted inthe port, the face of probe 185 and window 210 are substantiallyparallel. Port 205 and tip 235 are adapted such that the port and tipare reversibly connectable. In one embodiment, port 205 includes annularinset 214 and tip 235 includes opening 216 defined by annular lip 218for receiving inset 214. In this embodiment, inset 214 has a diameterless than opening 216. It will be appreciated that the connectingrelationship between the port and tip can be reversed (i.e., port havingannular lip for receiving tip having inset). Lip 218 defines bed 222 forreceiving substrate 220, which is secured in port assembly 202 when port205 is connected to tip 235. Tip 235 includes aperture 224 in bed 222.Aperture 224 provides for contact of substrate 220 with a liquid samplecontained in the sealed vessel.

Fluorescent species having pH-dependent emission. In one embodiment, thesystem and method for measuring pH uses a fluorescent species havingpH-dependent fluorescent emission. The fluorescent species has a firstemission intensity at a first wavelength and a second emission intensityat a second wavelength, the first and second emission intensities beingcharacteristic of pH in the environment of the fluorescent species. Theratio of the first and second emission intensities provides pHmeasurement. It is appreciated that fluorescent emission occurs as awavelength band having a band maximum that is referred to herein as theemission wavelength.

In one embodiment, the separation between the first wavelength and thesecond wavelength is at least about 40 nm. In one embodiment, theseparation between the first wavelength and the second wavelength is atleast about 30 nm. In one embodiment, the separation between the firstwavelength and the second wavelength is at least about 20 nm. Using 10nm HBW filters, the separation is at least about 30 nm. Preferably, thesystem achieves fluorescence signal separation by removing any emissionband overlap by 10E5 or more.

The system and method for measuring pH are not limited to any particularfluorescent species, nor any particular pH range. The system and methodare operable with any fluorescent species having pH-dependent propertiesthat can be excited and its emission measured. The range of pHmeasurable by the system and method can be selected and is determined bythe pH-dependent properties of the fluorescent species.

In addition to their pH-dependent properties noted above, suitablefluorescent species include those that can be substantially irreversiblyimmobilized on a substrate. The fluorescent species can be covalentlycoupled to the substrate or non-covalently associated with thesubstrate.

Suitable pH-dependent fluorescent species include those known in theart. Representative fluorescent species having suitable pH-dependentproperties include fluorescein derivatives including naphthofluoresceincompounds, seminaphthofluorescein compounds (e.g., SNAFL compounds), andseminaphthorhodafluor compounds (e.g., SNARF compounds). These compoundshave advantages associated with their long wavelength emission, which isless susceptible to potential interfering light absorbing substances inblood. These compounds also have relatively long wavelength absorbancemaking them particularly suitable for excitation by commerciallyavailable LED light sources. Another compound having suitable pHdependent behavior is HPTS, 8-hydroxy-1,3,6-pyrenetrisulfonic acid.Although the compound has desired ratiometric pH properties, excitationis optimal at short wavelength (403 nm) where strong LED light sourcesare not commercially available. Representative SNAFL and SNARF compoundsuseful in the system and method are described in U.S. Pat. No.4,945,171. Molecular Probes (now Invitrogen, Eugene, Oreg.) sells CNF,SNAFL, SNARF fluors with conjugatable carboxylic acid linker groups,see, for example, Molecular Probes Handbook (Ninth Edition) by R. P.Haugland, Chapter 21 “pH indicators” pages 829-847. Epoch Biosciences(now Nanogen, Bothell, Wash.) sells EBIO-3 with a propanoic acid linker.Whitaker et al. (Anal. Biochem. (1991) 194, 330-344) showed thesynthesis of a number of SNAFL compounds. Wolfbeis et al. (MikrochimActa (1992) 108, 133-141) described the use of CNF and aminocelluloseconjugates. The earliest reference to the SNAFL family of compounds isWhitaker et al. (1988) Biophys. J. 53, 197a. A related dye in the CNFfamily is VITABLUE, a sulfonenaphthofluorescein derivative (Lee et al.(1989) Cytometry 10, 151-164) having a pKa of 7.56. A CNF analog withbromine substituents at each carbon adjacent to a phenol (pKa 7.45) hasa pKa that is 0.54 pKa units lower than their measured pKa for CNF (pKa7.99). Lee et al. note that “true” pKa values are difficult to determinefor these compounds. A method for pKa determination is described inExample 12. SNAFL-1 (literature pKa ˜7.8) free acid had a pKa of 7.6 inthat fluorescence-based assay. Other suitable fluorescent speciesinclude the compounds described in U.S. Patent Application PublicationNo. US 2006/0204990 A1, published Sep. 14, 2006 (U.S. Ser. No.11/357,750).

The structures of seminaphthofluorescein compounds (SNAFL-1 and EBIO-3)useful in one embodiment of the system and method are illustrated below.

The numbering scheme describes position of attachment of linkermolecules. These compounds have carboxylate linking groups suitable forconjugation to carrier proteins, as described below. For conjugation,the reactive N-hydroxysuccinimide (NHS) ester of SNAFL-1 (commerciallyavailable from Molecule Probes, Inc., Eugene, Oreg.) can be used.Conjugation to lysine residues in human serum albumin (HSA) gave desiredSNAFL/HSA conjugates. Carbodiimide activation of EBIO-3 gave a reactiveintermediate that was efficiently conjugated to human serum albumin.

Representative seminaphthofluorescein compounds useful in a system andmethod are illustrated in FIGS. 23A-23E.

The SNAFL compounds are commercially available from Molecular Probes,Inc., Eugene, Oreg. The SNAFL compounds can be readily synthesizedaccording to general procedures that have been published (see, forexample, U.S. Pat. No. 4,945,171).

The preparation of a representative 2-chloro substituted SNAFL compoundis shown below.

The compound can be prepared by condensation of 1,6-dihydroxynaphthalenewith the diacid substituted 4-acylresorcinol in the presence of adehydrating acid or Lewis acid catalyst, such as zinc chloride.

The preparation of SNAFL compounds having propionic acid linkers isdescribed in U.S. patent application Ser. No. 11/022,039, incorporatedherein by reference in its entirety. A representative SNAFL compoundshaving a propionic acid linker, EBIO-3, is commercially available fromNanogen, Bothell Wash.

The emission spectra as a function of pH of representative fluorescentspecies (i.e., SNAFL-1 and EBIO-1) useful in the system and method areillustrated in FIGS. 24 and 25, respectively. FIG. 24 illustrates theemission spectra of SNAFL-1 in 50 mM potassium phosphate buffer as afunction of pH (pH 6.0 to 10.0) (excitation at 540 nm). Referring toFIG. 24, the response at pH 6-7 is relatively poor (pKa=7.6). FIG. 25illustrates the emission spectra of EBIO-3 in 50 mM potassium phosphatebuffer as a function of pH (pH 6.0 to 10.0) (excitation at 545 nm).Referring to FIG. 25, the response at pH 6-7 is relatively good(pKa=6.6). Spectral properties and pKa data for the SNAFL analogsillustrated in FIGS. 23A-23E are summarized in Table 1.

TABLE 1 pH-Sensitive absorbance and emission of SNAFL analogs. EmissionAbsorbance Absorbance Emission λmax Compound λmax (acid) λmax (base)λiso (base) pKa SNAFL-1 482, 510 nm 540 nm 585 nm 620 nm 7.6 SNAFL-2485, 514 547 590 630 7.6 EBIO-1 496, 519 545 560 620 6.5 EBIO-2 506, 538572 590 645 7.8 EBIO-3 480, 509 534 560 610 6.6

Referring to Table 1, absorbance and emission spectra were obtained at10 μM SNAFL analog. Absorbance was measured at pH 6, 8, and 10: acid (pH6) gave two bands of similar absorbance; pH 10 gave a single λmax(base). The emission spectra were determined by excitation at theabsorbance λmax (base). The wavelength where emission spectra crossed isreported as λiso. The emission λmax was measured at pH 10. pKa wasdetermined from fluorescence emission spectra. EBIO-1 and EBIO-3 weremore sensitive to changes at pH ˜6.5. The other analogs were moresensitive at pH ˜8.

Fluorescent species conjugates for substrate immobilization. For use ina system and method, the fluorescent species may be immobilized on asubstrate such that the fluorescent species is in contact with thesample, the pH of which is to be measured. The fluorescent species canbe immobilized on the substrate through the use of a material (e.g.,macromolecular spacer material) having a strong associative interactionwith the substrate. The spacer material allows covalent conjugation ofthe fluorescent species and provides large surface area needed forefficient non-covalent immobilization to the substrate surface. In oneembodiment, the spacer material is human serum albumin (HSA) having ˜44lysine residues available for covalent conjugation. HSA's denselycharged molecular structure has a passivating effect when adsorbed tobiomaterials. Other advantages include reduced fluorescence quenching,uniform environment for the conjugated fluorophore, and availability inrecombinant form (from yeast) so there is no chance of infection (aswith HSA from donors). HSA conjugates are easily purified byultrafiltration methods and form stable solutions that are easilycharacterized by absorbance and fluorescence assays to determine thenumber of fluorophores per protein.

In one embodiment, the fluorescent species is immobilized on thesubstrate through the use of a protein or protein fragment. Suitableproteins include those that can be substantially irreversiblyimmobilized on the substrate. The protein can be covalently coupled tothe substrate or non-covalently associated with the substrate. Suitableproteins include proteins to which the fluorescent species can besubstantially irreversibly immobilized. The fluorescent species can becovalently or non-covalently associated with the protein.

Suitable proteins include human serum albumin (HSA), bovine serumalbumin (BSA), vonWillebrand's factor, kininogen, fibrinogen, andhemoglobin (no iron). Suitable proteins include proteins havingavailable lysine residues (for conjugation to the fluorophore) andmolecular weight sufficient to allow for immobilization efficiency tothe blot membrane. Other functional groups in the protein (likecysteine) could presumably be used for covalent bonding to suitablyreactive solid supports.

In one embodiment, the fluorescent species is immobilized on thesubstrate through the use of a polysaccharide. Suitable polysaccharidesinclude those that can be substantially irreversibly immobilized on thesubstrate. The polysaccharide can be covalently coupled to the substrateor non-covalently associated with the substrate. Suitablepolysaccharides include proteins to which the fluorescent species can besubstantially irreversibly immobilized. The fluorescent species can becovalently or non-covalently associated with the polysaccharide.

Suitable polysaccharides include dextrans, aminodextrans, heparin, andlectins.

In another embodiment, the fluorescent species is immobilized on thesubstrate through the use of dendrimeric structures. Suitabledendrimeric structures include those that can be substantiallyirreversibly immobilized on the substrate. The dendrimeric structurescan be covalently coupled to the substrate or non-covalently associatedwith the substrate. PAMAM dendrimers are commercially available as areother structural types and sizes.

In one embodiment, the fluorescent species is covalently coupled to aprotein to provide a fluorophore-protein conjugate that can beimmobilized on a substrate. In one embodiment, thefluorophore-polysaccharide conjugate is non-covalently associated withthe substrate.

In one embodiment, a fluorophore-protein conjugate is immobilized on asubstrate. In one embodiment, the fluorescent species is aseminaphthofluorescein and the protein is human serum albumin. In oneembodiment, the seminaphthofluorescein is SNAFL-1. The preparation ofSNAFL-1/HSA conjugates is described in Example 13. The fluorescentproperties of SNAFL-1/HSA conjugates are described in Example 14. In oneembodiment, the seminaphthofluorescein is EBIO-3. The preparation ofEBIO-3/HSA conjugates is described in Example 1. A schematicillustration of the coupling of EBIO-3 to HSA is illustrated in FIG. 26.The fluorescent properties of EBIO-3/HSA conjugates are described inExample 15.

The fluorescent emission spectra as a function of pH (6.0 to 10.0) of arepresentative fluorophore-protein conjugate (SNAFL-1/HSA, 1.6fluorophores per HSA) useful in the system and method are illustrated inFIG. 27.

The fluorescent emission spectra as a function of pH (6.0 to 10.0) of arepresentative fluorophore-protein conjugate (EBIO-3/HSA, 1.92fluorophores per HSA) useful in the system and method are illustrated inFIG. 28.

For the fluorophore-protein conjugate, the optimum fluorophore loadingwill vary depending on the particular fluorophore.

For SNAFL-1/HSA conjugates the fluorophore loading can vary from about0.01 to about 38 SNAFL-1/HSA. Low signal at 0.01 and fluorescentquenching at 40 fluorophores/HSA. In one embodiment, the SNAFL-1conjugate includes about 2 SNAFL-1/HSA.

For EBIO-3/HSA conjugates the fluorophore loading can vary from about0.01 to about 40 EBIO-3/HSA. In one embodiment, the EBIO-3 conjugateincludes about 2 EBIO-3/HSA.

Substrates for fluorescent species immobilization. In the system andmethod described herein, the fluorescent species is immobilized on asubstrate. As noted above, the fluorescent species can be directlyimmobilized on the substrate covalently or by non-covalent associationor, alternatively, through the use of a material (e.g.,fluorophore-protein conjugate) that can be immobilized on the substratecovalently or by non-covalent association.

Suitable substrates substantially irreversible immobilized thefluorescent species. In the method, suitable substrates also do notinhibit the contact of the liquid sample with the fluorescent speciesand do not impair or alter the pH measurement.

Representative substrates include membranes, such as microporousmembranes made of cellulose, nitrocellulose, mixed esters ofnitrocellulose and cellulose acetate, polyethylene terephthalate,polycarbonate, polyvinylidene fluoride and polyimide. Such materials areavailable commercially from Whatman S&S, Florham Park, N.J. andMillipore, Billerica Mass. Suitable membranes include membranes in whichthe microporous structure is created by ion beam penetration such asmembranes commercially available from Oxyphen GmbH, Dresden, Germanyunder the designation OXYPHEN. Charged nylon surfaces (Nytran) can alsobe used. Suitable membranes include plastic membranes in which themicroporous structure is made by injection molding the micropores intothe plastic such as the processes used by Ømic, Stockholm, Sweden.Emission intensity of SNAFL-1/HSA at pH 7 immobilized on various poresize mixed ester nitrocellulose cellulose acetate membranes is shown inFIG. 38.

Immobilization of representative fluorophore protein conjugates onmembranes is described in Examples 16 and 2. Example 16 describes theimmobilization of SNAFL-1/HSA conjugates. Example 17 describes thefluorescent properties of immobilized SNAFL-1/HSA conjugates. Example 2describes the immobilization of EBIO-3/HSA conjugates. Example 18describes the fluorescent properties of immobilized EBIO-3/HSAconjugates.

The emission spectra of a representative fluorophore-protein conjugate(SNAFL-1/HSA, 3.6:1) immobilized on Oxyphen and nitrocellulose as afunction of pH (pH response), as measured by the microwell assaydescribed in Example 17, are illustrated in FIGS. 29 and 30,respectively.

The emission spectra of a representative fluorophore-protein conjugate(EBIO-3/HSA, 2.0:1) immobilized on nitrocellulose, as described inExample 16, as a function of pH (6.0, 6.5, 7.0, 7.5, 8.0, and 10.0), asmeasured by the telescoping tube insert assay described in Example 18,are illustrated in FIG. 31. The large spread of emissions at 600 nm forthe pH 6 to 8 range indicates good fluorescence verses pH response.

Ratiometric pH system and method. In one embodiment, the method is afluorescent wavelength-ratiometric method. In the method, the first andsecond fluorescent emission intensities of the fluorescent speciesmeasured at first and second emission wavelengths, respectively, areratioed to provide pH information. The first emission wavelength varieswith pH while the second emission wavelength is constant with pH andgives an internal control for the fluorescent intensity. In oneembodiment, a lookup table is used to lookup a combination of themeasured ratio, first and second emission wavelength and determines itscorresponding pH. In one embodiment, a mathematical function of theratio, first and second emission wavelength is used to calculate theresulting pH. FIG. 15 is a lookup table that correlates the ratio of thetwo emission intensities to pH. FIG. 16 is an example of data fromcalibration studies that are used to generate a lookup table such as theone in FIG. 15.

FIG. 32 illustrates the data used in the method for measuring pH. Theemission spectra of a representative fluorophore-protein conjugate(EBIO-3/HSA, 2:1) immobilized on nitrocellulose at pH 7.0 is shown asmeasured by the telescoping tubing insert assay. In this setup, theexcitation bandpass filter was unable to completely remove theexcitation light in the emission region as illustrated by the backgroundsignal measured on a blank nitrocellulose disc. The full spectrumcorrected for the background was multiplied by the transmittance of thefirst and second hypothetical filters at each wavelength and the areaunder the resultant curve was calculated to give a signal for the firstand second wavelength. The center wavelengths and bandwidths ofhypothetical filters were chosen such that the ratiometric properties ofthe conjugate had the strongest relationship to the pH in the region ofinterest.

FIG. 33 illustrates the results of the method for phosphate bufferedsaline (PBS), platelet poor plasma (PPP), and platelet rich plasma (PRP)as measured by the telescoping tubing insert assay described in Example18. The three curves represent the best fit relationship between themeasured pH and ratios for the three different liquids.

FIG. 34 illustrates the correlation of pH results for three differentplasma samples obtained by the system and method as measured by theinjection molded insert PVC tube assay described in Example 18. Therelationship between the fluorescent signal and the pH has an accuracyof about 0.1 pH units.

FIG. 35 illustrates stability of a representative substrate-immobilizedfluorophore conjugate (EBIO-3/HSA, 2:1) on mixed ester nitrocelluloseand cellulose acetate prepared by the soaking method and as measured bythe leaching assay described in Example 2. The low level of leaching isfar below the toxic level for any compound.

Carbon dioxide measurement. In another aspect, a device and method formeasuring carbon dioxide concentration in a liquid sample are provided.The carbon dioxide measuring method utilizes the pH measuring system andmethod described above. In the carbon dioxide measuring method anddevice, a substrate-immobilized fluorescent species as described aboveis in contact with a solution, the pH of which is responsive to carbondioxide level. In addition to being in contact with thesubstrate-immobilized fluorescent species, the solution having pHresponsive to carbon dioxide level is in contact with a liquid samplefor which the level of carbon dioxide is to be measured. The solutionhaving pH responsive to carbon dioxide level is isolated from the liquidsample for which the level of carbon dioxide is to be measured by aselectively permeable membrane. The membrane is permeable to gases(e.g., carbon dioxide) and impermeable to other materials (e.g.,liquids). Using the method of measuring pH described above, the pH ofthe solution responsive to carbon dioxide concentration in contact withthe substrate-immobilized fluorescent species is measured and correlatedwith the carbon dioxide level of the sample in contact with thatsolution.

The solution having pH response to carbon dioxide level is an aqueoussolution that includes an agent that is reactive toward carbon dioxideand changes the pH of the solution in response to carbon dioxideconcentration. Suitable agents that are reactive toward carbon dioxideand change the pH of the solution in which they are dissolved includebicarbonates, such as sodium bicarbonate.

The selectively permeable membrane isolates the solution having pHresponsive to carbon dioxide level from the liquid sample containingcarbon dioxide. The membrane is permeable to carbon dioxide andimpermeable to liquids and other solutes. In the method, carbon dioxidefrom the liquid sample passes from the liquid sample through themembrane and into the aqueous solution thereby reacting with the carbondioxide reactive agent to alter the pH of the aqueous solution. Suitableselectively permeable membranes include membranes made from silicone andPTFE.

FIG. 36 illustrates a representative device for measuring carbon dioxidein a sealed vessel. Referring to FIG. 36, device 600 includes portassembly 610 including port 620 and tip 630. Port 620 is a cylinderterminating with window 622 and having opening 624 for receiving probe185. When inserted in the port, the face of probe 185 and window 622 aresubstantially parallel. Port 620 and tip 630 are adapted such that theport and tip are reversibly connectable. Substrate 640 includingimmobilized fluorescent species is secured within port 620 and tip 630.Tip 630 includes a chamber 645 for receiving a solution having pHresponsiveness to carbon dioxide. Chamber 645 is defined by window 622,tip 630, and selectively permeable membrane 650. Chamber 645 includessubstrate 640, which is interrogated by probe 185.

A device for measuring carbon dioxide was assembled as described abovewith the membrane containing immobilized EBIO-3/rHSA conjugate (rHSA isrecombinant HSA). A layer of PARAFILM M, a blend of olefin-typematerials, was added under the membrane towards the tip. The membranewas hydrated with 5 ul of 35 mM carbonate buffer (pH 7.4), which wassealed within the assembly by the PARAFILM M and remained hydratedthroughout the assay. The assembly was subjected to 100% carbon dioxidegas by connection to the gas source with tubing and a “Y” adapter tobleed off the pressure. The assembly was subjected to the carbon dioxidefor an allotted period of time, allowed to acclimate to ambient airconditions, and repeated. The fluorescence was measured at each stage at568 nm and 600 nm after being excited at 525 nm. The results aresummarized below in Table 2 and reflect changes in fluorescence due tothe change in carbon dioxide concentration demonstrating that thefluorometric ratio method can also be used to calculate carbon dioxideconcentration. The PVC storage bags that are used for platelet storageare somewhat gas permeable, and carbon dioxide is directly related tothe measurement of pH.

TABLE 2 Carbon dioxide sensing results. Emission at Emission at RatioEnvironmental Conditions 568 nm 600 nm (600/568) 15 min. at ambient CO₂753 2184 2.9  5 min. at 100% CO₂ 1179 2234 1.894 15 min. at ambient CO₂833 2175 2.611  8 min. at 100% CO₂ 1161 1930 1.662 60 min. at ambientCO₂ 765 2184 2.854

In one embodiment, a fluorescence-based pH indicator that can be easilyinserted into the sampling ports of designed blood storage bags andinterrogated using a fiber optic-based LED light source and photodiodemeasurement system is provided. In one embodiment, this solid statesystem uses a “ratiometric” calibration method that accounts forvariability in fluorescent signal strength due to interfering substancesin blood that may interfere with the amount of excitation light thathits the indicator dye. The ratio of fluorescence intensities aremeasured at two wavelengths, one to detect the acid (protonated) isomerof the dye and one to detect the base (deprotonated) isomer.

To develop an accurate pH detector for platelet rich plasma, compoundshaving pKa of ˜6.6 are suitable, for example, 2-chloro substitution ofSNAFL compound lowers the pKa of the phenol from 7.6 to ˜6.6. Conjugatesof these compounds can be immobilized to various solid supports toprovide sensing pH membranes.

In one embodiment, an inexpensive, easy to manufacture pH sensingmembrane that gives accurate measurement of pH in platelet storage bagsat pH 6.5-7.5 is provided. In one embodiment, a protein conjugate (humanserum albumin) of a 2-chloro substituted ratiometric fluorescentcompound is used. The fluorophore:HSA ratio was optimized forperformance when immobilized to a nitrocellulose blot membrane. Afterdrying on the membrane, the fluorophore:HSA conjugate has very lowleaching rates. Discs of this material are easily assembled into holdersfor insertion into the sampling ports of platelet storage bags. Thefluorescent membrane materials showed good pH response using a green LEDbased fluorometer. In the method, two emission wavelengths forratiometric pH detection are measured with properly filtered photodiodeswith an accuracy of ˜0.1 units at the desired low pH threshold of 6.5.

Fluorescent probe molecules can be designed to be sensitive to a varietyof environments. The system and method describe the use of pH-sensitivefluorophores. However, other environments can be interrogated by thesystem and method modified to include environment-sensitive fluorophoresother than pH-sensitive fluorophores. A variety of fluorescent probesthat change fluorescent properties as the molecular environment changesare commercially available. See, for example, Molecular Probes Handbook(9^(th) Ed.) by R. P. Haugland. Probes can be linked to albumins orother proteins and used to prepare substrates for interrogation asdescribed in herein or using other fluorescent-based methods. Examplesof environment-sensitive fluorophores, systems, and methods include thefollowing.

Nucleic acid detection: nucleic acid binding dyes change fluorescentproperties in the presence of DNA or RNA.

Enzyme substrates: proteins or peptides can be labeled with fluorescentdyes and fluorescent quenching molecules such that a fluorescent signalis generated in the presence of particular enzymes such as proteases(FRET detection).

Probes for lipids: lipophilic dyes can change fluorescent properties inthe presence of cell membranes or other lipid rich analytes.

Probes for oxygen: in addition to pH detection and carbon dioxidedetection, certain fluorescent molecules are sensitive to changes inoxygen concentration, for example, tris(2,2′-bipyridiyl)ruthenium(II)dichloride (RTDP).

Indicators for metal ions: fluorescent dyes that bind metals can changefluorescent properties upon binding calcium, magnesium, zinc, sodium,potassium, among others.

Glucose detection: certain lectins such as ConA bind glucose, andsuitably labeled lectins can be prepared as probes for glucose.

One object is to at least measure the parameters described above inorder to solve some of the problems associated with platelet storage insealed sterile containers. There are several problems or disadvantagesdescribed hereinafter that may be solved by the current invention asmultiple parameters are monitored over time within the environment ofthe sealed platelet storage device. The additional advantages, objects,or computational features of the invention described herein will becomeapparent to those having some skill in the art upon examination orpractice of the invention. The advantages and objects of the inventionmay be attained as particularly pointed out in the appended claims.

In one aspect, the invention provides methods and apparatus for testingthe sterility of a product to be administered to a patient. The methodsand apparatus provide for the determination of the sterility of theproduct by determining whether bacteria is present in the product.

Transfusion of bacterially contaminated platelet concentrates isassociated with significant morbidity and mortality. A leading cause ofserious adverse events related to transfusions is bacterial sepsis.Microbial monitoring is being implemented by blood donation services toreduce the risk of transfusion-associated morbidity and mortality causedby contaminated blood components. International pharmacopoeias requirecomplete sterility of pharmaceutical products that are injected into apatient's blood stream or that otherwise enter the body below the skin'ssurface. Manufacturers of such products are required to supply proof ofsterility of the final product batch.

In practice, closed systems such as STERISART® NF are used for sterilitytesting of pharmaceutical products. The STERISART® system is a membranefilter system that eliminates the manipulation of filters therebyavoiding a main risk of secondary contamination and eliminating falsepositives resulting from secondary contamination. In these systems, asample is pumped into the filter device and filtered through thedevice's membrane, the membrane collecting any bacteria present in thesample. After rinsing, culture medium is added to the device and thedevice including culture medium is incubated without contact with theenvironment. Bacteria and/or spores filtered from the sample andcollected on the membrane filter are cultured in the medium. Growth canbe accelerated by storage of the medium-containing filter devices atelevated temperature. After some time, bacterial growth causes themedium to become cloudy in appearance (McFarland standard). Cloudinessbecomes visible when the bacteria concentration is sufficiently high(about 10E7 bacteria per mL medium). However, depending on the amount ofbacteria present in the sample and the rapidity of bacterial growth inthe culture medium, the time required for cultured medium to becomecloudy can be significant.

Because bacteria growth in a medium results in pH change and because themethods and apparatus of the invention can detect small changes in pH,the methods and apparatus of the invention can be used to detect thepresence of bacteria in culture medium long before the appearance ofcloudiness.

In the methods of the invention, a product to be administered to apatient is filtered through a membrane filter device. Any bacteriapresent in the product is collected on the membrane through which theliquid product is filtered. Once the product has been filtered throughthe device, culture medium is added to the device. In the presence ofthe medium, the membrane-collected bacteria grows. The growth ofbacteria in the device results in a pH change of the medium (pHdecrease), which is detected by the sensor.

The pH change of the medium is measured by the fluorescent methoddescribed herein. In the method, the sensor is in liquid communicationwith the culture medium and the medium pH is monitored. A pH decrease isindicative of the presence of bacteria in the original product. No pHchange is indicative of the absence of bacteria in the original product(i.e., the original product is sterile).

In one aspect, the invention provides a method for testing the sterilityof a sample. In one embodiment, the method includes:

(a) providing a filter device having a chamber and a filter configuredto collect culturable organisms;

(b) filtering a sample through the filter such that any collectedculturable organisms are retained in the chamber;

(c) adding a culture medium to the chamber;

(d) irradiating a fluorescent species immobilized on a substrate withexcitation light emanating from a probe physically isolated from thefluorescent species immobilized on the substrate,

wherein the fluorescent species immobilized on the substrate is inliquid communication with the culture medium,

wherein the excitation light has a wavelength sufficient to effectfluorescent emission from the fluorescent species, and

wherein the fluorescent species exhibits a first emission intensity at afirst emission wavelength and a second emission intensity at a secondemission wavelength, the ratio of the first and second emissionintensities being dependent on pH;

(e) measuring the first and second emission intensities to determine thepH of the culture medium; and

(f) inferring from the determined pH of the culture medium the sterilityof the sample.

In one embodiment, the filter includes a membrane having a pore size ofabout 45 μm.

In one embodiment, the probe is physically isolated from the fluorescentspecies immobilized on the substrate by a window transparent to theexcitation light and the fluorescent emission. In one embodiment, theprobe includes one or more optical fibers.

In one embodiment, the fluorescent species is selected from the groupconsisting of a naphthofluorescein compound and a seminaphthorhodaminecompound. In one embodiment, the fluorescent species is anaphthofluorescein compound selected from the group consisting of aseminaphthofluorescein compound and a carboxynaphthofluoresceincompound.

In one embodiment, the fluorescent species is a seminaphthofluoresceincompound selected from the group consisting of 5′(and6′)carboxy-3,10-dihydroxy-spiro[7H-benzo[c]xanthene-7,1′(3′H)-isobenzofuran]-3′-oneand2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoicacid.

In one embodiment, the fluorescent species is a conjugate of afluorescent compound and a macromolecule. In one embodiment, themacromolecule is an albumin. In one embodiment, the fluorescent speciesis a seminaphthofluorescein/human serum albumin conjugate.

In one embodiment, the sample is blood or a blood product.

In another aspect, the invention provides a method for testing thesterility of a sample. In one embodiment, the method includes:

(a) providing a filter device having a chamber and a filter configuredto collect culturable organisms;

(b) filtering a sample through the filter such that any collectedculturable organisms are retained in the chamber;

(c) adding a culture medium to the chamber;

(d) irradiating a fluorescent species in communication with the culturemedium with excitation light having a wavelength sufficient to effectfluorescent emission from the fluorescent species,

wherein the fluorescent species exhibits a first emission intensity at afirst emission wavelength and a second emission intensity at a secondemission wavelength, the ratio of the first and second emissionintensities being dependent on pH, and

wherein the fluorescent species is a 2-halo seminaphthofluorescein;

(e) measuring the first and second emission intensities to determine thepH of the culture medium; and

(f) inferring from the determined pH of the culture medium the sterilityof the sample.

In one embodiment, the 2-halo seminaphthofluorescein is a 2 chloroseminaphthofluorescein. In one embodiment, the 2-haloseminaphthofluorescein is2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoicacid.

In one embodiment, the fluorescent species is a conjugate of a 2-haloseminaphthofluorescein and a macromolecule. In one embodiment, themacromolecule is an albumin. In one embodiment, the macromolecule is ahuman serum albumin. In one embodiment, the conjugate is a2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoic acid/human serum albumin conjugate.

In one embodiment, the sample is blood or a blood product.

The apparatus of the invention useful for sterility testing are membranefilter devices that include a sensor capable of detecting pH changes inthe medium contained in the device. A change in pH is indicative of thepresence of bacteria and the lack of sterility of the product.

In another aspect, the invention provides an apparatus for testing thesterility of a sample liquid. In one embodiment, the apparatus includes:

(a) a chamber having an inlet and an outlet;

(b) a filter disposed at the chamber outlet, the filter adapted tocollect culturable organisms from a liquid passed through the filter;and

(c) a port assembly having a distal end extending into the chamber, thedistal end having a fluorescent species immobilized on a substrate,

wherein the fluorescent species is in fluid communication with aninterior of the chamber,

wherein the fluorescent species is a material that, when irradiated withan excitation light having a wavelength sufficient to effect fluorescentemission from the fluorescent species, exhibits a first emissionintensity at a first emission wavelength and a second emission intensityat a second emission wavelength, the ratio of the first and secondemission intensities being dependant on pH, and

wherein the port assembly further includes a housing for receiving aprobe.

In one embodiment, the filter includes a membrane having a pore size ofabout 45 μm.

In one embodiment, the housing comprises an open first end and a secondend terminating with a window.

In one embodiment, the port assembly includes a tip member reversiblyconnectable to the housing second end, wherein the tip member is adaptedto receive liquid from a sample to be tested.

In one embodiment, the apparatus includes:

(a) a light source for exciting the fluorescent species;

(b) a first emission detector for measuring the first emissionintensity;

(c) a second emission detector for measuring the second emissionintensity;

(d) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the excitationlightguide has a first terminus proximate to the light source and asecond terminus distal to the light source;

(e) a first emission lightguide for transmitting emission from thefluorescent species to the first emission detector, wherein the firstemission lightguide has a first terminus proximate to the first emissiondetector and a second terminus;

(f) a second emission lightguide for transmitting emission from thefluorescent species to the second emission detector, wherein the secondemission lightguide has a first terminus proximate to the secondemission detector and a second terminus; and

(g) a probe housing, wherein the probe housing houses the second terminiof the excitation lightguide, first emission lightguide, and secondemission lightguide.

In one embodiment, the light source is a light-emitting diode. In oneembodiment, the first and second emission detectors are photodiodes. Inone embodiment, the excitation lightguide, the first emissionlightguide, and the second emission lightguide are optical fibers.

In one embodiment, the port assembly includes a tapered tube terminatingwith the window.

In one embodiment, the fluorescent species is aseminaphthofluorescein/human serum albumin conjugate.

Representative apparatus of the invention are illustrated in FIGS. 39Aand 39B.

Referring to FIG. 39A, apparatus 1100 includes inlet 1102 forintroducing liquid into the device and membrane filter 1110 intermediatefirst chamber 1106 and second chamber 1108. Chamber 1108 is openallowing for the exit of liquid introduced into first chamber 1106. Portassembly 232 (see FIG. 22 for port assembly components) allows forinterrogation of liquids (e.g., culture medium) in first chamber 1106 asdiscussed below.

Referring to FIG. 39B, apparatus 1100 a includes inlet 1102 forintroducing liquid into the device, membrane filter 1110 intermediatefirst chamber 1106 and second chamber 1108, and outlet 1109 a forexiting liquid introduced into first chamber 1106. Port assembly 232(see FIG. 22 for port assembly components) allows for interrogation ofliquids (e.g., culture medium) in first chamber 1106.

In another aspect, the invention provides an apparatus for testing thesterility of a liquid sample. In one embodiment, the apparatus includes:

(a) a chamber having an inlet and an outlet;

(b) a filter disposed at the chamber outlet, the filter adapted tocollect culturable organisms from a liquid passed through the filter,the filter having a fluorescent species immobilized thereon,

wherein the fluorescent species is in fluid communication with aninterior of the chamber,

wherein the fluorescent species is a material that, when irradiated withan excitation light having a wavelength sufficient to effect fluorescentemission from the fluorescent species, exhibits a first emissionintensity at a first emission wavelength and a second emission intensityat a second emission wavelength, the ratio of the first and secondemission intensities being dependant on pH; and

(c) a port assembly having a distal end adjacent to the filter, the portassembly further including a housing for receiving a probe.

In one embodiment, the filter includes a membrane having a pore size ofabout 45 μm.

In one embodiment, the housing comprises an open first end and a secondend terminating with a window.

In one embodiment, the apparatus includes:

(a) a light source for exciting the fluorescent species;

(b) a first emission detector for measuring the first emissionintensity;

(c) a second emission detector for measuring the second emissionintensity;

(d) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the excitationlightguide has a first terminus proximate to the light source and asecond terminus distal to the light source;

(e) a first emission lightguide for transmitting emission from thefluorescent species to the first emission detector, wherein the firstemission lightguide has a first terminus proximate to the first emissiondetector and a second terminus;

(f) a second emission lightguide for transmitting emission from thefluorescent species to the second emission detector, wherein the secondemission lightguide has a first terminus proximate to the secondemission detector and a second terminus; and

(g) a probe housing, wherein the probe housing houses the second terminiof the excitation lightguide, first emission lightguide, and secondemission lightguide.

In one embodiment, the light source is a light-emitting diode. In oneembodiment, the first and second emission detectors are photodiodes. Inone embodiment, the excitation lightguide, the first emissionlightguide, and the second emission lightguide are optical fibers.

In one embodiment, the port assembly includes a tapered tube terminatingwith the window.

In one embodiment, the fluorescent species is aseminaphthofluorescein/human serum albumin conjugate.

Representative apparatus of the invention are illustrated in FIGS. 40Aand 40B.

Referring to FIG. 40A, apparatus 1200 includes inlet 1202 forintroducing liquid into the device and membrane filter 1210 intermediatefirst chamber 1206 and second chamber 1208. Chamber 1208 is openallowing for the exit of liquid introduced into first chamber 1206. Portassembly 232 b allows for interrogation of liquids (e.g., culturemedium) in first chamber 1206. It is contemplated that in thisembodiment, port assembly 232 b does not include substrate 220 and ortip 235.

Referring to FIG. 40B, apparatus 1200 a includes inlet 1202 forintroducing liquid into the device, membrane filter 1210 intermediatefirst chamber 1206 and second chamber 1208, and outlet 1209 a forexiting liquid introduced into first chamber 1206. Port assembly 232 b(see FIG. 22 for port assembly components) allows for interrogation ofliquids (e.g., culture medium) in first chamber 1206. It is contemplatedthat in this embodiment, port assembly 232 b does not include substrate220 and or tip 235.

In the apparatus of the invention, the membrane filter (e.g., 1110 and1210) effectively collects culturable organisms such as bacteria andspores from a sample filtered through the membrane. The membrane has apore size sufficiently large so as to readily pass the sample andsufficiently small so as to collect bacteria and spores that may becontained in the sample (e.g., 0.45 μm membrane).

To determine the presence of culturable organisms collected by themembrane, culture medium is introduced into the first chamber of thedevice and, optionally, incubated (e.g., 37° C.). The culture medium isthen monitored over time for the presence of bacteria by monitoring thepH of the medium.

Port assembly 232 allows for interrogation of the pH of the culturemedium introduced to the device's first chamber with probe 185 asdescribed above. Port assembly 232 receives probe 185, which transmitsexcitation light from the excitation source to the fluorescent speciesin liquid communication with the culture medium and which transmitslight emitted from the fluorescent species to the emission detector.

In certain embodiments, port assembly 232 is positioned in the firstchamber 1106 wall (see FIGS. 39A and 39B). In these embodiments, culturemedium is introduced into the device at a level such that the portassembly 232 is in liquid communication with the medium.

In certain other embodiments, port assembly 232 b is positioned in thesecond chamber 1208, 1208 b adjacent the membrane 1210 (see FIGS. 40A,40B, and 41). Referring to FIG. 41, port assembly 232 b with associatedprobe 185 interrogates the culture medium in the first chamber 1206 byinterrogating membrane 1210. In this embodiment, the fluorescent speciesis immobilized on at least a portion of membrane 1210. In thisembodiment, port assembly 232 b does not include substrate 220 and ortip 235, and window 210 is positioned adjacent the portion of membrane1210 to which the fluorescent species is immobilized.

In certain embodiments, the invention provides a system forsimultaneously evaluating multiple samples (see, for example, FIGS. 42and 43). In these embodiments, a plurality of apparatus of the inventionare positioned in the devices having a plurality of stations, eachstation receiving an apparatus of the invention. Each station alsoprovides a probe for interrogating culture medium in the filter device,and each filter device port assembly receives a probe.

In one aspect the invention provides a system for testing the sterilityof a liquid sample. In one embodiment, the system includes:

(a) a plurality of stations, each station configured to receive aculture assembly;

(b) a plurality of culture assemblies, each culture assembly including:

-   -   (i) a chamber having an inlet and an outlet;    -   (ii) a filter disposed at the chamber outlet, the filter adapted        to collect culturable organisms from a liquid passed through the        filter;    -   (iii) a port assembly having a distal end extending into the        chamber, the distal end having a fluorescent species immobilized        on a substrate,

wherein the fluorescent species is in fluid communication with aninterior of the chamber,

wherein the fluorescent species is a material that, when irradiated withan excitation light having a wavelength sufficient to effect fluorescentemission from the fluorescent species, exhibits a first emissionintensity at a first emission wavelength and a second emission intensityat a second emission wavelength, the ratio of the first and secondemission intensities being dependant on pH; and

wherein the port assembly further includes a housing for receiving aprobe.

In one embodiment, each apparatus includes:

(a) a light source for exciting the fluorescent species;

(b) a first emission detector for measuring the first emissionintensity;

(c) a second emission detector for measuring the second emissionintensity;

(d) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the excitationlightguide has a first terminus proximate to the light source and asecond terminus distal to the light source;

(e) a first emission lightguide for transmitting emission from thefluorescent species to the first emission detector, wherein the firstemission lightguide has a first terminus proximate to the first emissiondetector and a second terminus;

(f) a second emission lightguide for transmitting emission from thefluorescent species to the second emission detector, wherein the secondemission lightguide has a first terminus proximate to the secondemission detector and a second terminus; and

(g) a probe housing, wherein the probe housing houses the second terminiof the excitation lightguide, first emission lightguide, and secondemission lightguide.

In another aspect, the invention provides a system for testing thesterility of a liquid sample. In one embodiment, the system includes:

(a) a plurality of stations, each station configured to receive aculture assembly;

(b) a plurality of culture assemblies, each apparatus including:

-   -   (i) a chamber having an inlet and an outlet;    -   (ii) a filter disposed at the chamber outlet, the filter adapted        to collect culturable organisms from a liquid passed through the        filter, the filter having a fluorescent species immobilized        thereon,

wherein the fluorescent species is in fluid communication with aninterior of the chamber,

wherein the fluorescent species is a material that, when irradiated withan excitation light having a wavelength sufficient to effect fluorescentemission from the fluorescent species, exhibits a first emissionintensity at a first emission wavelength and a second emission intensityat a second emission wavelength, the ratio of the first and secondemission intensities being dependant on pH; and

-   -   (iii) a port assembly having a distal end adjacent to the        filter, the port assembly further including a housing for        receiving a probe.

In one embodiment, each apparatus includes:

(a) a light source for exciting the fluorescent species;

(b) a first emission detector for measuring the first emissionintensity;

(c) a second emission detector for measuring the second emissionintensity;

(d) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the excitationlightguide has a first terminus proximate to the light source and asecond terminus distal to the light source;

(e) a first emission lightguide for transmitting emission from thefluorescent species to the first emission detector, wherein the firstemission lightguide has a first terminus proximate to the first emissiondetector and a second terminus;

(f) a second emission lightguide for transmitting emission from thefluorescent species to the second emission detector, wherein the secondemission lightguide has a first terminus proximate to the secondemission detector and a second terminus; and

(g) a probe housing, wherein the probe housing houses the second terminiof the excitation lightguide, first emission lightguide, and secondemission lightguide.

Referring to FIG. 42, system 1301 includes a plurality of stations 1310for receiving culture assembly 1100. Culture assembly 1100 receiveprobes 185 via port assemblies 232. Referring to FIG. 43, system 1400includes a plurality of stations 1410 for receiving culture assembly1200. Culture assemblies 1200 receive probes 185 via port assemblies 232b, as described above. The filter devices can be positioned for analysiseither before or after addition of culture medium to the devices. FIG.44 illustrates representative apparatus of the invention positioned foranalysis. Referring to FIG. 44, first apparatus 1200 is positionedbefore addition of culture medium and second apparatus 1200 ispositioned after addition of culture medium 1250 (or culture medium 1250is added to apparatus 1200 after positioning).

Liquid (e.g., sample and culture medium) can be introduced to theapparatus of the invention by pumping liquid into the apparatus. Liquidcan be removed from the apparatus by pumping or by exerting partialvacuum. Liquid can be removed from the apparatus while positioned instations (see FIGS. 42-44) through an aperture (see 1320 in FIG. 42 and1420 in FIGS. 41 and 43). Liquid is conducted away from the apparatusthrough a conduit (see, for example, the conduit illustrated in FIG.44).

Another representative apparatus of the invention is illustrated in FIG.45. Referring to FIG. 45, apparatus 1500 includes septum 1502, outlet1504, and membrane filter 1510 intermediate first chamber 1506 andsecond chamber 1508. Port assembly 232 b allows for interrogation ofliquids (e.g., culture medium) in first chamber 1506. In thisembodiment, the fluorescent species is immobilized on at least a portionof membrane filter 1510. In this embodiment, port assembly 232 b doesnot include substrate 220 and or tip 235, and window 210 is positionedadjacent the portion of membrane filter 1510 to which the fluorescentspecies is immobilized. Port assembly 232 b receives probe 185. Liquidscan be introduced into the apparatus through septum 1502 pierced withsyringe assemblies 1520 and 1522 that can be connected to liquidreservoirs (e.g., sample, aerobic culture medium, anaerobic culturemedium). Multiple apparatus 1500 can be evaluated simultaneously throughthe use of multiple probes 185 in a manner similar to those illustratedin FIGS. 42 and 43.

The following examples are provided for the purposes of illustrating,not limiting, the invention.

Examples Example 1 The Preparation of Fluorophore-Protein Conjugates:EBIO-3/HSA

Method A. A 0.1 M stock solution of EDC (Sigma/Aldrich Chemical Co., St.Louis Mo.) was prepared by dissolving 6.2 mg of EDC in 0.2 mL of DMF and0.123 mL of 50 mM phosphate buffer (pH 5.8). 1.0 mg of EBIO-3 acid(Nanogen, Bothell, Wash.) was dissolved in 0.102 mL of DMF to give a 20mM solution. 3.0 mg (0.045 micromoles) of HSA (Sigma/Aldrich ChemicalCo., St. Louis, Mo.) was dissolved in 0.3 mL of pH 8.5 sodiumbicarbonate in each of two 1.7 mL Eppendorf tubes. 0.1M EDC (0.045 mL)was added to 20 mM EBIO-3 (0.045 mL, 0.9 micromoles) in a separateEppendorf tube and this was added to one of the HSA tubes to give anEBIO-3:HSA offering ratio of 20:1. An offering ratio of 5:1 was used inthe other HSA tube by adding a premixed solution of 0.0225 mL of EDC(0.1 mM) and 0.0225 mL of EBIO-3 (20 mM). The homogeneous dark red HSAconjugate solutions were incubated at room temperature in the dark.After 21 hours, each of the HSA conjugates was purified on a G15Sephadex column as described above for the SNAFL conjugates (Example13). Some unreacted EBIO-3 acid remained at the top of the column(especially for the 20:1 offering ratio), but was cleanly separated fromthe desired protein conjugate that eluted first as a pink fraction in˜0.5 mL of pH 7.4 buffer. Each of the purified conjugates was diluted to0.75 mL with pH 7.4 PBS to give 4 mg/mL solutions (0.06 mM). The redsolutions were stored refrigerated and protected from light. 1micromolar solutions of each EBIO-3/HSA conjugate were prepared at pH7.4 and analyzed by UV-vis spectra using a Beckman DU640B spectrometer.The free EBIO-3 acid (10 micromolar) spectrum had absorbance maximum at534 nm, the 20:1 conjugate had absorbance at 538 nm and the 5:1conjugate had maximum at 545 nm. The spectra showed the expectedincrease in absorbance with increasing EBIO-3:HSA offering ratio. Usingthis EBIO-3 acid as a standard, the 20:1 conjugate had 5.07 EBIO-3:HSAand the 5:1 offering had 1.92 EBIO-3:HSA. The coupling efficiency wassomewhat lower than for the SNAFL/HSA conjugates of Example 4 (the 20:1conjugate had 11.2 fluors/HSA and the 5:1 offering had 4.1 fluors/HAS).The EDC coupling method was suitably efficient and reproducible.

Method B. A 0.1 M solution of EDC (Sigma/Aldrich Chemical Co., St.Louis, Mo.) is prepared by dissolving 6.0 mg of EDC in 0.194 mL of DMFand 0.118 mL of 50 mM PBS (pH 7.4). 3.0 mg of EBIO-3 acid (Nanogen,Bothell, Wash.) is dissolved in 0.306 mL of DMF to give a 20 mMsolution. The two solutions are combined in the EBIO-3 solutioncontainer and incubated at room temperature for one hour in the dark.75.0 mg (1 micromole) of liquid recombinant HSA (rHSA) from yeast (DeltaBiotechnology, Ltd., Nottingham, UK) is mixed with 7.5 mL of pH 8.5sodium bicarbonate in a 15 mL conical tube. The entire contents of theEBIO-3/EDC solution are combined with the rHSA solution and incubated atroom temperature in the dark for 15-20 hours. The rHSA/EBIO-3 conjugateis purified using the Amicon stirred ultrafiltration cell system and aYM10 membrane (Millipore, Bedford, Mass.). A 50 mM PBS (pH 7.4) is usedas the wash solution. After purification, the protein concentration ofthe conjugate is measured using the BCA™ Protein Assay (Pierce,Rockford, Ill.). An aliquot of the conjugate is diluted to 1 mg/mL with50 mM PBS (pH 7.4) based on its BCA determined protein concentration.The 1 mg/mL aliquot of conjugate, the last milliliter of PBS effluent,an aliquot of the 50 mM PBS (pH 7.4), and an aliquot of the EB3 Standard(15 mM EBIO-3 solution in DMF and 50 mM PBS (pH 7.4)) are analyzed viaan absorbance scan utilizing Bio-Tek's Synergy HT plate reader. The scanis taken on 300 microliters of each of the above mentioned samples in ablack, 96-well, clear, flat bottom plate, scanned from 450 nm to 650 nm.Their max peaks are recorded and used to determine purity and quality ofthe conjugate.

Example 2 Immobilization of Fluorophore-Protein Conjugates: EBIO-3/HSA

Spotting Immobilization Method. EBIO-3/HSA conjugate was prepared asdescribed in Example 1 at a ratio of 2:1. Nitrocellulose membranes wereobtained from Schleicher and Schuell under the trade name PROTRAN. Thediscs were treated in the same way as the general immobilization methoddescribed in Example 14 using a 4 mg/mL solution of EBIO-3/HSA.

Soaking Immobilization Method. EBIO-3/HSA conjugate was prepared asdescribed in Example 1 at a ratio of 2:1. Mixed ester nitrocellulose andcellulose acetate membranes were obtained from Millipore under theproduct series TF. The EBIO-3/HSA conjugate is diluted to 0.2 mg/mL and45 mL is added to a 9 cm disc of the membrane. The disc is agitatedovernight at room temperature and protected from light. The unboundconjugate is removed and the disc is washed with two 1 hour washes andone overnight wash all with agitation. The disc is then desiccated andstored dry. Smaller discs are punched from the 9 cm disc for studies.

Example 3 The Manufacture of a Vessel Incorporating aSubstrate-Immobilized Fluorescent Species

PVC material is compounded with a number of additives, for example,plasticizers, stabilizers, and lubricants. The formulation is used formaking bags and tubes. The compounded PVC is extruded through a die orcalendared in a press for converting the plasticized material into sheetform. The extruded sheet, after slitting, is cut into the desired sizeand sent to the welding section. The donor and transfer tubings are madeby extrusion of similar PVC compounds. The tubes are then cut to theappropriate length and sent to the welding section. The components, suchas transfusion ports, needle covers, and clamp, are produced byinjection molding. The components are ultrasonically cleaned and driedin a drying oven.

Welding. The blood bags are fabricated by a high frequency weldingtechnique. Sized PVC sheets are placed between electrodes and highfrequency at high voltage is applied. PVC gets heated very rapidly andsealing takes place between electrodes. Transfusion ports and donor andtransfer tubing are kept in the appropriate position with the bag andwelded to form an integral part of the blood bag system. For themanufacture of a vessel incorporating a representativesubstrate-immobilized fluorescent species, an open tube is welded toprovide port 510A. The tube can be made of colored PVC to provide lightprotection for the immobilized fluorescent species. Welded bags aretrimmed. The port assembly 232 (FIG. 22) is manufactured from injectionmolded Lexan parts (205 and 235) and a 3.53 mm ( 9/64 inch) diameternitrocellulose disc with immobilized fluorescent species (220). The portassembly is held together by friction fit or can be glued in place. Theport assembly is inserted in the open tube of port 510A. The portassembly is held in the port by friction fit or can be glued in place.The assembled bag and port assembly is sterilized and labeled forultimate storage of platelet concentrates.

Example 4 The Incorporation of a Substrate-Immobilized FluorescentSpecies into a Sealed Vessel

The port assembly is manufactured from injection molded Lexan parts anda 3.53 mm ( 9/64 inch) diameter nitrocellulose disc with immobilizedfluorescent species. The port assembly is held together by friction fitor can be glued in place. The port assembly is inserted through theseptum seal inside port 510A by puncturing the seal with the spiked tip.Alternatively, the seal can be pre-punctured with a separate spike tool.The insertion of the port assembly can be performed on either empty orplatelet filled bags, but in either case, aseptic methods should be usedto avoid possible contamination of the bag contents. The port assemblyis held in the port by friction fit or can be glued in place. The vesselremains sealed (leakproof) after insertion of the port assembly in theport.

Example 5 Assembly of Inserts and Bags

The methods described in Examples 3 and 4, above, were followed with thefollowing exceptions: an altered version of the injection molded inserttip 235, shown in FIG. 22, was used. After friction fit assembly, theshaft, tip and membrane disc were pushed into an opaque blue sleeve ofPVC tubing (Natvar, City of Industry, Calif., durometer 80 Shore A,dimensions=ID 0.216″, OD 0.291″, L 1.15″) until the flange contacted thetubing. The prongs just reach the other end of the tubing. This frictionfit assembly was fit inside the assembled large bag as shown in FIG. 1.

Assembly of inserts into bags. The bags were welded with large diameterPVC tubing (inner diameter slightly smaller than the outer diameter ofthe blue insert assembly, ¼ inch shorter than the blue insert assembly).The blue insert assembly was held into the larger diameter PVC tube inthe bag by friction fit or by solvent welding with cyclohexanone. Theblue sleeve protrudes from the flush cut bag tubing by ¼ inch, thusproviding a space for the “clip” on the instrument.

Preparation of Small Bags.

Method A. The small bag shape shown in FIG. 2 was welded from previouslyassembled large bags (with no inserts or twist off sample ports). One ofthe tubing ports for the sample ports is discarded along with much ofthe citrate PVC film. The blue pH reading insert assembly, pigtailtubing and sample ports were carefully assembled with cyclohexanonesolvent welding and packaged for sterilization.

Method B. Two sheets of breathable citrate PVC film (Solvey-Draka) areRF welded together using a steel tool that contacts the film in theouter shape of the bag in FIG. 2. PVC tubes on steel rods are alsoplaced between the two layers of the film before the RF welding. Thisforms a leak proof seal with the tubes and film. After the RF weldingthe steel rods are removed so that twist off ports, pigtails and sensorscan be placed in the PVC tubes. These features are solvent bonded orfixed in another way into the tubes.

Ethylene Oxide Sterilization of bag/insert assembly. The bags wereindividually wrapped and sterilized using a standard ethylene oxidecycle used for other platelet storage bags. The overwrapped bags werepackaged in boxes and unwrapped just prior to use.

Example 6 Protocol Description for Large Bag pH and Platelet Health

An in vitro evaluation of PCs during storage in representative large andsmall platelet storage containers with integrated pH probes wasperformed. The evaluation determined that the integrated pH device hadno negative impacts on in vitro quality of platelets during storage,either during storage in standard platelet containers or during storagein down-scaled containers for research purposes. The following examplerefers to the data presented in FIGS. 3-7.

Platelet Storage Containers

A. 4 large experimental PVC-citrate storage containers (lot numberm4340) with integrated pH probe (for storage of about 300 mL PC), ETOsterilized.

B. 4 large approved PVC-citrate storage containers (brand Fresenius,T2110, batch H10211011) (for storage of about 300 mL PC), steamsterilized.

C. 4 small experimental PVC-citrate containers with integrated pH probe(for storage of about 17 mL PC), ETO sterilized.

D. 4 small PVC-DEHP (so-called buffy coat bags; 100-150 mL nominalvolume), steam sterilized.

All other materials are standard materials used by the Sanquin BloodCentre Region North West for blood collection and by the SR-BTTlaboratory for blood processing and testing.

A representative system (serial number PH05100007, firmware version2.00, Blood Cell Storage, Inc., Seattle, Wash.) was used fornon-invasive fluorescence measurements of the storage containers withintegrated pH probes. For the data presented in FIGS. 3-10 and 16, eachbag was positioned on a representative system (device) and thefluorescent ratio was determined from which pH was calculated.Readings/samplings were done at 4 hours post set-up then every 6-12hours later over a period of 7 to 11 days. The volume in the smallstorage containers was to about 15 mL.

The first two PC were combined, and then two small bags were filled witheach about 15 mL, followed by distribution of the remaining volume intwo equal volumes over the two types of large storage containers.

In order to ascertain a good starting quality of the platelets, ameasurement of CD62P expression on day 1 of storage was included.

The number of platelets positive for PAC-1 (fibrinogen binding site onglycoprotein IIb/IIIa) was not determined, because the test was shown tobe influenced by the presence of plasma.

Blood Collection

Standard blood collection systems (manufactured by Fresenius HemoCare,Emmer-Compascuum, the Netherlands) were filled with approximately 500 mLof blood during blood collection at the Sanquin Blood Centre RegionNorthWest (Sanquin Blood Supply Foundation, Amsterdam, the Netherlands)from non-enumerated, informed donors. The day of blood collection wasdesignated as day 0 of the study.

Blood collections were performed under standard conditions, with the aidof calibrated blood collection balances equipped with a mixing platformallowing mixing at regular intervals, monitoring of blood flow andbleeding time, a final check of the weight of the donation and coolingof the blood to 20° C. immediately after collection. At least 40donations (20 from A-Rh(+) and 20 from 0-Rh(+) donors) meeting thecriteria of volume (500±50 mL of blood) and bleeding time (<15 min) wereselected by the Blood Centre for further processing to plasma, red cellconcentrate in SAG-Mannitol and buffy coat 12 to 16 h after the bloodcollection (on day 1 of the study). After centrifugation and Compomat G4separation of the whole blood, at least 40 buffy coats were delivered tothe BTT laboratory. In addition, per set of 5 buffy coats a plasma unitfrom one of the corresponding 5 donations was delivered.

Preparation of Pooled Platelet Concentrates

The 40 buffy coats were used to prepare 6 pools, each consisting of 5buffy coats and one unit of plasma. Pools consist of units of the sameblood group. From these buffy coat pools, platelet concentrates wereprepared after a second centrifugation step (Hettich Roto Silenta/RP, 5min at 1700 rpm, 905×g, brake 3). Conditions for these separations areselected in such a way that from each pool of buffy coats a plateletconcentrate (PC) of ±320 mL volume with 1-1.5×10e9 platelets/mL and aleukocyte contamination of less than 50×10e7 leukocytes per concentratewas obtained. During preparation of PC from the pooled buffy coats, theconcentrates are filtered over a Compostop CS leukoreduction filter(Fresenius Hemocare, 50 cm², T3995) with a large PVC-citrate containerconnected to the outlet of the filter. For 4 PC this container wascontainer A and for 4 PC this was container B (see Materials andInstruments; A and B were matched for blood group).

Pooling and Splicing of Platelet Concentrates

After preparation, a PC in container A was combined with a PC incontainer B (blood group matched) and after mixing two times 15 mL wastransferred to a container C and a container D. Subsequently, theremaining volume was redistributed over the A and B containers in such away that the volume was equal.

Storage of Platelet Concentrates

The filtered PC were stored for 8 days after preparation at 22° C.±2° C.horizontally shaking with 1 cycle per minute. Samples were taken underaseptic conditions after 1, 2, 3, 6, 7 and 8 days of storage (meaningday 2, 3, 4, 7, 8 and 9 of PC shelf-life) and analyzed for variousplatelet quality parameters.

Measurements

The following features of platelet quality were measured:

1. Morphological parameters (measured after 3 days, 6 days and 8 days ofstorage). The morphological parameters involve the screening of theswirling (resulting in a swirling score) and the microscopiccharacterization of the different forms of platelets, i.e. discoid,discoid with dendrites, balloons or spheres, resulting in a so-calledKunicki score.

2. Physical changes (measured after preparation and after 1, 2, 3, 6, 7and 8 days). The physical changes are characterized by measurements ofthe pH, P(O₂) and P(CO₂) and the number of leukocytes (counted at day 0)and the number of platelets.

3. Changes in activation degree (measured after 3 days, 6 days and 8days). The changes in degree of activation were measured with amonoclonal antibody, directed against activation-dependent antigenCD62P. Also the percentage of platelets expressing phosphatidyl-serine(PS, measured with AnnexinV) was determined.

4. Metabolic changes (measured after 3 days, 6 days and 8 days). Themetabolic changes are characterized by the intracellular amount ofadenine nucleotides, the extracellular concentration of glucose andlactate and the mitochondrial membrane potential (as measured withJC-1).

At the end of the storage period, the platelet concentrates in the largecontainers were checked for sterility.

A. Composition of the Platelet Concentrates.

During preparation of the platelet concentrates from the pooled buffycoats, no deviations from normal procedures were observed, with afiltration time over the in-line filter of about 6 min. The eightleukodepleted products were used to prepare 4 pools, which weresubsequently distributed over the small containers (type C and D, each15 mL) and the large containers (type A and B, about 300 mL each). Table2 shows that the PC in type A and type B containers were very similar.After leukoreduction, the number of leukocytes was well below 1×10⁶ inall units (as determined by fluorescent Nageotte counting, with forevery sample 0 counted). The total number of platelets in each poolexceeded 300×10⁹, and these numbers meet the requirements of the Councilof Europe for leukocyte depleted PC.

TABLE 3 Composition of the platelet concentrates Type A Type B Volume PCmL 313 ± 5.8  308 ± 6.2  Platelet conc. ×10⁹/mL 1172 ± 58.0  1172 ±58.0  Total platelets ×10⁹ 367 ± 16.8 361 ± 18.3 Total WBC ×10⁶ 0.10 ±0.08  0.09 ± 0.07  Values given are the mean ± SD of 4 concentrates.

B. Change in Morphological Parameters.

The viability of platelets after transfusion correlates fairly well withplatelet morphology. The most discoid platelets have the best in vivosurvival, whereas spherical platelets perform much less. A morphologicalindex was introduced by Kunicki to predict viability of platelets aftertransfusion. The number of discs identified in a 100 cell count of fixedplatelets under the microscope is multiplied by 4, spheres by 2,platelets with dendrites by 1, and balloons by 0, resulting in a maximalscore of 400 for perfect discoid platelets.

The morphology scores and the percentage discoid platelets duringstorage of the PC pools are depicted in Table 2.

TABLE 4 Morphological parameters of platelet concentrates during storageDay 4 Day 7 Day 9 of shelf-life of shelf-life of shelf-life Bag Kunicki% Kunicki % Kunicki % type score discoid score discoid score discoid AMean 295 50 279 44 248 31 SD 20.4 8.2 11.8 4.8 8.7 2.5 B Mean 288 46 26338 228 24 SD 22.5 9.5 15.5 6.5 8.7 2.5 C Mean nd nd 268 39 nd nd SD ndnd 5.0 2.5 nd nd D Mean nd nd 263 36 Nd nd SD nd nd 17.1 8.5 Nd ndValues given are the mean ± SD of 4 concentrates. Days indicate thenumber of days after blood collection (day 0). nd: not determined.

The first measurement, after 3 days of storage (day 4 of PC shelf-lifeindicated in the table), indicates a high quality of the platelets.During further storage the morphology score remains high, with onlyminimal differences between the two types of large containers tested.Also the values for the small containers, only determined on day 7, werevery similar, indicating also good storage characteristics for thesesmall containers.

Another method to judge morphology of platelets is the observation ofthe swirling pattern in the storage bag. The “silk-like” patterns areobserved visually, after brief squeezing of the bag and the degree ofinhomogeneity is scored according to the following scale:

3. Swirling inhomogeneity throughout the whole bag, with contrastobservable as fine detail

2. Swirling inhomogeneity visible throughout the whole bag with goodcontrast

1. Some inhomogeneity visible, but only in a few places and with poorcontrast

0. Turbid homogeneity, no effect of squeezing.

With this method for freshly prepared platelet concentrates a score isobtained of 3, whereas after storage for 6 days the score should beabove 2.

Table 2A shows that during storage for up to 8 days (day 9 ofshelf-life) the score in the large containers was above 2, with minimaldifferences between the two tested types of container. In the smallcontainers the score was above 2 till day 6, whereas the swirling scorewas slightly lower in the small PVC-DEHP bag compared to the smallPVC-citrate bag.

TABLE 5 Swirling scores of platelet concentrates during storage Type day2 day 4 day 7 day 8 day 9 Mean A 3.0 2.6 2.5 2.4 2.1 SD 0.0 0.1 0.0 0.30.3 Mean B 3.0 2.8 2.2 2.2 2.3 SD 0.0 0.2 0.3 0.1 0.2 Mean C 3.0 2.8 2.42.4 2.2 SD 3.0 0.2 0.1 0.1 0.1 Mean D 3.0 2.9 2.0 1.9 1.6 SD 3.0 0.1 0.20.1 0.1 Values given are the mean ± SD of 4 concentrates. Days indicatethe number of days after blood collection (day 0). nd: not determined.

C. Change in Physical Parameters.

When during storage of platelets the pH falls below 6.7 (measured at 37°C.), disc-to-sphere transformation occurs and morphology of theplatelets becomes worse. This change becomes irreversible when the pHfalls below 6.5 and therefore, at the end of storage, CE requirementsprescribe a pH between 6.6 and 7.2 (measured at 37° C.) or between 6.8and 7.4 (measured at 22° C.). The pH values during storage in the largecontainers type A and B and the values for pCO₂, pO₂ and bicarbonate aregiven in Table 3a. During storage the pH remains above 6.9 during 8 days(=9 days of shelf-life), with only minimal differences between the twocontainer types tested. In the small containers (type C and D) the pHwas only measured on day 6 of storage. Compared to the large containersthe pH was slightly lower on this day, indicating slightly worse storageconditions in the small containers. pCO₂ and pO₂ showed the normalpattern during storage of platelet concentrates.

TABLE 6 Physical parameters during storage pH at 37° C. pCO₂ pO₂ HCO₃ ⁻Shelf-life type mean SD Mean SD mean SD mean SD Day 1 A 7.058 0.023 66.52.1 156.0 2.7 18.3 0.5 B 7.058 0.023 66.5 2.1 156.0 2.7 18.3 0.5 Day 2 A7.196 0.013 43.8 0.5 80.6 6.6 16.6 0.5 B 7.173 0.008 46.7 0.5 65.8 10.516.8 0.4 Day 3 A 7.229 0.019 36.5 1.8 87.4 7.3 14.9 0.7 B 7.200 0.00639.1 1.6 73.2 4.7 14.9 0.7 Day 4 A 7.229 0.010 33.4 0.6 84.3 8.8 13.60.4 B 7.190 0.008 36.2 2.1 65.6 5.2 13.5 0.6 Day 7 A 7.095 0.018 30.21.5 98.4 6.1 9.1 0.7 B 7.077 0.010 32.1 1.7 84.6 5.6 9.2 0.3 Day 7 C7.052 0.089 19.5 1.3 153.4 7.7 0.8 5.4 D 6.900 0.054 41.1 3.1 117.5 13.21.0 7.9 Day 8 A 7.013 0.025 29.1 1.2 110.2 10.3 7.3 0.6 B 7.004 0.00631.3 0.9 91.3 4.1 7.6 0.2 Day 9 A 6.921 0.027 30.1 1.7 107.4 4.5 6.1 0.7B 6.935 0.007 31.4 0.8 90.0 4.2 6.5 0.2 Values given are the mean ± SDof 4 concentrates. Days indicate the number of days after bloodcollection (day 0).

During storage in the large containers the platelet concentration showedonly minimal variation during storage, whereas for the small containersabout 5% decrease was found at day 7 (Table 3b).

TABLE 7 Platelet concentration during storage bag type day 1 day 3 day 4day 7 day 8 Mean A 1172 1215 1221 1219 1233 SD 58.0 68.9 55.1 67.1 79.3Mean B 1202 1226 1250 1182 SD 69.1 65.5 64.9 46.0 Mean C 1163 SD 109.0Mean D 1137 SD 83.3 Values given are the mean ± SD of 4 concentrates.Days indicate the number of days after blood collection (day 0).

D. Changes in Metabolic Parameters During Storage.

The glucose concentration measured after 1 day of storage (day 2 of PCshelf-life) was similar as observed in most other studies with storagein plasma.

Glucose consumption during storage was also similar as previouslyobserved (about 6 mM in 5 days) with a concomitant increase of lactateof about 10 mM, indicating a major role for aerobic glycolysis inglucose consumption. Hardly any differences were found between thetested types of large containers. The values for the small containerswere also very similar, but the results indicated slightly more glucoseconsumption in the small containers compared to the large containers.

TABLE 8 Glucose and lactate in supernatants of PC's during storage Day 12 3 4 7 8 9 Bag A glucose 20.7 20.0 18.8 17.3 14.1 13.7 10.4 lactate D6.6 7.9 10.1 10.2 17.0 18.1 19.1 Bag B glucose 20.7 20.3 19.2 17.7 14.314.2 11.0 lactate D 6.6 8.3 10.4 10.2 16.3 17.3 17.4 Bag C glucose 20.712.1 lactate D 6.6 21.3 Bag D glucose 20.7 13.1 lactate D 6.6 18.3Values given are the mean ± SD of 4 concentrates, paired study.

The metabolic state of platelets is well characterized by theintracellular amounts of nucleotides on the various days. A correlationbetween ATP content and in vitro survival was found by Holme et al.Platelets contain two pools of nucleotides, a metabolic pool with anATP:ADP ratio of about 10, and a storage pool (in the dense granules)with an ATP:ADP ratio of about 1, resulting in an overall ratio of about2 in a total platelet extract. It was found in earlier studies thatduring storage the storage pool is depleted in favor of the metabolicpool, reflected by a slightly increased ATP/ADP ratio.

The results for the nucleotide analyses (Table 9) showed no differencesbetween the two tested containers and overall the results are similar toresults found with other studies using the B-type container at SR-BTT.The results for the small containers at day 7 (not shown) were verysimilar to the data found at day 7 for the large containers.

TABLE 9 Nucleotide content in PC's during storage units day 4 day 7 day9 A-series ATP pmol/10⁶ 51.4 ± 1.22 46.4 ± 4.83 41.9 ± 1.72 ADP pmol/10⁶27.7 ± 1.00 22.7 ± 2.25 19.2 ± 0.86 AMP pmol/10⁶  4.3 ± 0.31  3.1 ± 0.45 2.8 ± 0.20 ATP:ADP 1.85 ± 0.02 2.04 ± 0.01 2.19 ± 0.09 B-series ATPpmol/10⁶ 51.2 ± 4.04 45.1 ± 1.37 42.1 ± 2.00 ADP pmol/10⁶ 27.4 ± 2.6121.4 ± 1.04 18.4 ± 0.86 AMP pmol/10⁶  3.5 ± 0.32  3.6 ± 1.20  2.6 ± 0.24ATP:ADP 1.87 ± 0.03 2.11 ± 0.06 2.28 ± 0.07 Values given are the mean ±SD of 4 concentrates, paired study.

The mitochondrial membrane potential (Δψ) was measured with thefluorescent dye JC-1. This dye accumulates in the mitochondrial matrixin response to the membrane potential across the mitochondrial innermembrane and, above a critical concentration, J-aggregates are formedwhich show a red shift in emission characteristics as compared to themonomeric state. Therefore, the ratio of red (FL2) over green (FL1)fluorescence as measured in the flow cytometer is an indicator for theΔψ, with a high value indicating active mitochondria and a lower valueindicative for mitochondrial damage. Table 10 shows that the FL2/FL1ratio starts high on day 2 and remains high during storage, with onlyminimal differences between the two types of containers tested.

E. Change in Membrane Characteristics of the Platelets.

Changes in cell surface antigens are indicative for the degree ofactivation of platelets during storage of PC's at room temperature.These so-called activation antigens can be detected with monoclonalantibodies (mAb). In this study we used an antibody against CD62P. Inresting platelets, the CD62P antigen is only present in the membranes ofα-granules, whereas after activation this antigen is also expressed onthe cell membrane.

Results for Large Containers

Table 10 shows the changes in expression (percentage positive cells) forCD62P from day 2 to day 9 of the PC shelf-life. The results show amoderate degree of activation, as is observed in all studies on plateletstorage, whereas there are minimal differences between the two types oflarge containers tested.

TABLE 10 Changes in cell surface markers and JC-1 during storage of PC'sCD62P AnnexinV JC-1 Series mean % SD mean % SD mean ratio SD Day 2 B 7.61.0 nd Nd Day 4 A 11.2 1.4 7.3 0.9 2.9 0.5 Day 4 B 10.9 1.4 6.6 0.9 3.00.3 Day 7 A 13.2 1.4 14.2 1.6 3.2 0.3 Day 7 B 12.5 1.9 14.8 1.2 3.0 0.2Day 9 A 19.9 2.0 16.7 2.2 3.0 0.2 Day 9 B 16.7 1.7 17.3 1.6 2.8 0.2Values given are the mean ± SD of 4 concentrates (paired study); %:percentage positive platelets.

In addition to the measurement of the cell surface expression of theCD62P antigen, the percentage of cells expressing phosphatidyl-serine(PS) was measured. Under normal conditions platelets exhibit anasymmetric distribution of phospholipids in the membrane. Thecholine-containing phospholipids, phosphatidyl choline andsphingomyelin, predominantly reside in the outer leaflet, while theaminophospholipids, phosphatidyl-ethanolamine (PE) and PS, are foundmainly in the inner leaflet. PS-exposure can be measured with Annexin-Vand a high expression is generally thought to be a signal for removal ofcells from the circulation. Table 6a shows the percentage of plateletspositive for Annexin-V binding on the various days during storage,indicating that initially a very low percentage of cells are positivefor PS-exposure. During storage some increase is found, but the meanvalue at day 9 is still low, indicating a good in vitro quality.Moreover, the values found for the two types of container tested arevery similar in the paired study.

Results for Small Containers

Table 11 shows the changes in expression (percentage positive cells) forCD62P from day 2 to day 7 of the PC shelf-life for the two types ofsmall containers tested. The results show a moderate degree ofactivation, slightly higher than that observed on day 7 in the largecontainers. Only minimal differences were seen between the smallPVC-citrate bag with a representative probe (type C) and the smallPVC-DEHP bag (type D). The JC-1 ratio on day 7 was similar to that foundin the large containers.

TABLE 11 Changes in cell surface markers and JC-1 during storage of PC'sCD62P AnnexinV JC-1 Series mean % SD Mean % SD mean ratio SD Day 2 B11.6 14.4 Nd nd Day 7 C 17.6 1.5 16.8 1.6 3.2 0.3 Day 7 D 18.5 1.2 16.33.8 3.6 0.5 Mean ± SD for n = 4, paired study.

F. Sterility at the of the Storage Period.

At the end of the storage period of 14 days (after day 9 somenon-invasive fluorescent measurements were performed, separatelyreported), one PC (2A) was found to be positive after direct seeding insoybean-casein hydrolysate or fluid thioglycolate as substrate andincubation at 20-25° C. respectively 30-32° C., according to CLBprotocol M 139 (title “Onderzoek op steriliteit”). Upon looking back tothe individual data there was no reason to exclude 2A from the data set,most probably this was a contamination introduced in the final days ofstorage, due to the frequent sampling for blood gasses (normally thesample site coupler is used 3-4 times during a study, this study up to10 times).

Conclusions

The platelet concentrates met the requirements of the research protocoland those of the European guidelines.

During storage under standard blood bank conditions, only minimaldifferences were found between the storage in the representativecontainer compared to the control Fresenius container (both PVC-citrate,paired study with full-scale platelet concentrates in plasma).

During storage under standard blood bank conditions, the variousmeasured in vitro parameters for both types of large containers tested,were similar to values observed in earlier platelet storage studies.

The values for the in vitro quality parameters at the end of study (8days of storage, 9 days of shelf-life) predicted a good in vivo recoveryand survival at the end of maximal (7 days) shelf-life.

Overall, the results of the present study indicate no harmful effectinduced by the tested representative platelet storage containers withintegrated pH probe.

The analysis of in vitro parameters on day 6 of storage in the smallcontainers indicated that the storage conditions for the down-scaledapproach were slightly worse than in the full-scale study. Therefore, itwould be better to reduce the stored volume in the small containers to13-14 mL, in order to mimic the full-scale conditions more closely.

Example 7

Bacterial Strains. The data in FIGS. 9 and 10 were obtained by measuringvarious parameters of PC samples inoculated with bacteria. Bacterialstrains were cultivated from −80° C. frozen stocks by two consecutiveovernight passages at 37° C. on 5% blood agar (PML, Wilsonville, Oreg.).All strains used were received as Microbiologics Kwik-Stiks purchasedfrom PML except the Escherichia coli strain (CFT073) which was receivedfrom Dr. S. Mosely at the University of Washington, Seattle, Wash. TheAmerican Type Culture Collection strains used are Staphylococcus aureusATCC 29213, Klebsiella oxytoca ATCC 43863, K. pneumoniae ATCC 13882,Serratia marcescens ATCC 43861 and Pseudomonas aeruginosa ATCC 27853.All strains except the E. coli were conditioned in plasma over threeserial passages with incubation at 22° C. than frozen at −80° C.

Processing and inoculation of bacterial strains. The small plateletstorage bag depicted in FIG. 2 was used for the bag tracking studies. Asampling port (Baxter Healthcare Corp., Deerfield, Ill.) is placed ineach bag. Using a 20 mL syringe (Becton Dickinson ((BD)), FranklinLakes, N.J.) and a 22G needle (BD), approximately 13-14 mL of plateletconcentrate (PC) is removed from the apheresis unit received from PugetSound Blood Center and aseptically transferred to the small bag for useas a normal or spiked sample. Underfilled samples were prepared withonly 7 mL of PC. The inoculum for the spiked bags is obtained from anovernight subculture on blood agar of the desired strain at 37° C.without CO₂. A suspension of bacteria in sterile saline is adjusted tothe turbidity of a 0.5 McFarland Standard which is roughly equivalent to1E8 CFU/mL. This suspension is further diluted in sterile saline andspiked into the prepared bags to give a final concentration ofapproximately 10, 100 or 1000 CFU/mL.

Sampling for Analysis by Bayer Blood Gas Analyzer and One-Touch UltraGlucose Meter. Following transfer of PC and spiking of bacteria, smallbags were placed in the Helmer incubator/shaker at 22° C. for 4 hours ofequilibration. After initial equilibration the bags were removed, thesampling port was cleansed with 95% ethanol and a sample was removedusing a 1 mL syringe (BD) with 22G needle. The sample was dividedbetween culture for CFU/mL, glucose meter and blood gas analyzer (BayerRapidLab model number 348. Bacterial spiked bags required a sample of˜0.4 mL for testing. Bags presumed to be sterile (normal or underfilled)required a sample of ˜0.25 mL for testing as sterility checks were doneinstead of CFU/mL determinations.

CFU/mL determinations were performed by serial dilution (1 in 10) of 0.1mL platelets. The amount plated on Tryptic Soy Agar (PML) was 0.1 mLplatelets (and/or dilutions thereof), utilizing ˜0.2 mL of sample.Plates were incubated at 37° C., colonies were enumerated after 24 hoursand CFU/mL were calculated. The limit of quantitation is 10 CFU/mL. Onedrop of PC on sheep blood agar was used as a sterility check with theplates incubated at 37° C. for at least 4 days.

Approximately 0.15 mL of the sample was used for pH blood gas analysis(Bayer 348, Bayer HealthCare, Norwood, Mass.). Samples were runimmediately after dispensing the amount necessary for CFU/mL orsterility determinations. Readings for pH, pCO₂ and pO₂ were obtained.

Glucose was determined utilizing the OneTouch Ultra meter and teststrips (Lifescan, Milpitas, Calif.) following manufacturer'sinstructions. Sample utilized per reading was less than 10 μl andresults were recorded in mg/dL.

Example 8 Fluorescence and pH Properties of SNAFL Analogs: pKaDetermination

Instrumentation. Fluorescence versus pH of various SNAFL free acids werecompared using an Ocean Optics USB2000 fiber optic spectrometer and atungsten halogen light source (part number HL-2000 FHSA). The lightsource was equipped with a linear variable filter that allowed thewavelength and shape of the excitation beam to be adjusted. Theexcitation wavelength was adjusted by using a blank cuvette to theabsorbance max of the fluorophore (see Table 1). A cuvette holder (partnumber CUV-FL-DA) was directly attached to the light source and a fiberoptic cable directed emitted light to the spectrometer. Excitationconditions are reported for each fluorescence spectrum (3000 msecirradiation at the indicated wavelength). Spectral data were collectedon a personal computer using the Ocean Optics software and overlays ofdifferent spectra were captured.

Sample preparation. SNAFL-1 was purchased as the free carboxylic acidfrom Molecular Probes in a 1 mg vial. 0.3 mL of isopropyl alcohol and0.7 mL of water was added to make a 1 mg/mL solution. A molecular weight(MW) of 426 for SNAFL-1 was used to calculate molarity (SNAFL-1=2.35mM). 4.25 uL of this solution was diluted to 1 mL with various 50 mMphosphate buffers to give 10 micromolar solutions with pH 6-10. 10micromolar solutions of SNAFL-2 (MW=460) were prepared in a similarfashion. EBIO-1 (MW=523), EBIO-2 (MW=627), and EBIO-3 (MW=489) wereobtained as bulk compounds from Epoch Biosciences. 1.6 mg of each solidpowder was carefully weighed out and dissolved in 3.2 mL of 40%isopropyl alcohol to give 0.5 mg/mL solutions. Emission spectra wereobtained for the various SNAFL and EBIO compounds at pH 6.0, 6.2, 6.4,6.6, 6.8, 7.0, 8.0 and 10.0. Examples of overlayed fluorescence emissionspectra are shown in FIG. 26 (SNAFL-1) and FIG. 27 (EBIO-3). All spectrashowed an isosbestic wavelength where all emission spectra overlap (SeeTable 1). This is a characteristic of ideal ratiometric performance withno competing fluorescent structures other than those shown above(lactone, naphthol, naphtholate).

pKa calculations. The pH at which two molecular species (tautomers) areequally represented is defined as the pKa. There are many variables thatcan affect pKa and methods for measurement are difficult since thestructures have overlapping absorbance. Therefore direct comparisonsfrom the literature can vary slightly. The calculations contained hereinare based on the assumption that, at pH 10, only the trianionicnaphtholate structure is present. The intensity of fluorescence at theemission maxima is divided by 2, and pH of the intersecting pH curve iscalculated by interpolation between the nearest 2 curves. The pKa of the2-chloro substituted EBIO compounds is significantly lower than theother analogs as shown in Table 1.

Example 9 The Preparation of Fluorophore-Protein Conjugates: SNAFL-1/HSA

Human serum albumin (HSA) was purchased from Sigma (catalog #A-8763) as100 mg of lyophilized powder. SNAFL-1 NHS ester was purchased fromMolecular Probes as a mixture of the 5 and 6 isomers. A solution of 10mg (0.15 micromoles) of HSA in 1 mL of pH 8.56 sodium bicarbonate (0.1M) was prepared. A solution of 1 mL (1.91 micromoles) of the NHS esterin 0.1 mL of dimethylsulfoxide was prepared. 0.3 mL aliquots of the HSAsolution were transferred to a 1.6 mL Eppendorf tubes and variousoffering ratios of the NHS ester solution were added: tube 1, 11.8microliters (5 equivalents); tube 2, 23.6 microliters (10 equivalents),tube 3, 47.1 microliters (20 equivalents). The deep red solutions werevortexed and allowed to stand in the dark for at least one hour. The 5:1conjugate from tube 1 was purified by gel filtration chromatography on a0.5×20 cm column packed with Sephadex G-15 and pH 7.4 phosphate bufferedsaline (PBS). The conjugate was isolated as a fast moving red/orangeband in PBS and diluted to 0.75 mL with PBS to give a 4 mg/mL solutionof the protein conjugate. Most of the color eluted with the conjugate,but some small molecular weight (orange) impurities remained on top ofthe column. The column was clean enough to be re-used for purificationof the 10:1 and 20:1 conjugates. Each was eluted in PBS and diluted to0.75 mL to give ˜4 mg/mL solutions (60 micromolar based on HSAcomponent). The red solutions were stored refrigerated and protectedfrom light. 1 micromolar solutions of each SNAFL-1/HSA conjugate wereprepared and analyzed by UV-vis spectra using a Beckman DU640Bspectrometer. Each spectrum showed absorbance maxima at 490 and 521 nmat pH 7 as expected for the acid form of SNAFL-1 conjugates. Therelative absorbance showed the expected change in absorbance withdifferent SNAFL:HSA offering ratio. A 10 micromolar solution of SNAFL-1acid (obtained from Molecular Probes) at pH 7 was used as a standard tomore accurately determine the average loading of SNAFL-1 per each HSAconjugate preparation. Using this assay, the 5:1 conjugate had 4.1fluors/HSA, the 10:1 conjugate had 6.4 fluors/HSA, and the 20:1conjugate had 11.2 fluors/HSA.

Example 10 The Fluorescent Properties of Fluorophore-Protein Conjugates:SNAFL-1/HSA

Relative fluorescence of various SNAFL-1/HSA conjugates and SNAFL-1 freeacid were compared using an Ocean Optics USB2000 fiber opticspectrometer and a tungsten halogen light source (part number HL-2000FHSA). The light source was equipped with a linear variable filter thatallowed the wavelength and shape of the excitation beam to be adjusted.A cuvette holder (part number CUV-FL-DA) was directly attached to thelight source and a fiber optic cable directed emitted light to thespectrometer. Excitation conditions are reported for each fluorescencespectrum (3000 msec irradiation at the indicated wavelength). Spectraldata were collected on a personal computer using the Ocean Opticssoftware and overlays of different spectra were captured. A comparisonof various loading levels of SNAFL-1/HSA showed that 4.1 to 1.6 SNAFL-1molecules gave about the same fluorescent signal. Higher loading orlower loading conjugates gave lower signals.

Emission spectra were obtained for 10 micromolar solutions in potassiumphosphate buffer. Excitation was at 540 nm. Emission maximum at 620 nmwas observed for the base form of SNAFL-1 (pH 10). As expected,intensity of 620 nm fluorescence decreased as pH decreased. Anisosbestic point at 585 nm, where fluorescence remained constant at allpH, was observed. Response was good at about pH 8, but poor between pH6-7.

Spectra obtained for a 2.5 micromolar solution of a representativeSNAFL-1/HSA conjugate (1.6 SNAFL-1/HSA) showed improved pH response forpH 6-7 (see FIG. 29). The Ocean Optics halogen light source was equippedwith a 532 nm interference filter (Edmund Optics, Barrington, N.J.) andthis allowed the fluorescent isosbestic point at 572 nm for pH 6-7 to bedetected. Emission maximum at 620 nm was observed for the base form ofSNAFL-1 (see pH 10 curve). As expected, intensity of 620 nm fluorescencedecreased as pH decreased. In comparison to the free SNAFL-1 carboxylicacid (see FIG. 26) improved response for pH 6-7 was observed for the HSAconjugate. A red shift of the pH 8 and 10 curves from the isosbesticwavelength was observed, indicative of other competing molecularstructures involving the fluorescent species. This non-ideal behaviormay be eliminated by use of a longer linker structure or a morehydrophilic linker structure between the fluorescent dye and the HSAspacer.

Example 11 The Fluorescent Properties of Fluorophore-Protein Conjugates:EBIO-3/HSA

Fluorescence spectra were obtained for 2.5 micromolar solutions of thetwo EBIO-3/HSA conjugates prepared as described in Example 1 (Method A).The conjugates showed improved pH response for pH 6-7 (see FIG. 12 foroverlayed spectra for the 1.92:1 EBIO-3/HSA conjugate). The Ocean Opticshalogen light source was equipped with a 532 nm bandpass filter (EdmundOptics, Barrington N.J.) and this allowed the fluorescent isosbesticpoint at ˜565 nm for pH 6-7 to be detected. Emission maximum at 605 nmwas observed for the base form of SNAFL-1 (red trace, pH 10). Asexpected, intensity of 605 nm fluorescence decreased as pH decreased. Incomparison to the SNAFL-1/HSA conjugate (see FIG. 29) improved responsefor pH 6-7 was observed for the EBIO-3/HSA conjugate. A red shift of thepH 8 and 10 curves from the isosbestic wavelength was observed,indicative of other competing molecular structures involving thefluorescent species, but was of smaller magnitude than for theSNAFL-1/HSA conjugate.

Example 12 Immobilization of Fluorophore-Protein Conjugates: SNAFL-1/HSA

Fluorophore-protein conjugates and fluorophore-carbohydrate conjugateswere immobilized on either nitrocellulose or capillary pore membranesusing the following general method. Fluorescein labeled dextrans with“fixable” lysine residues were obtained from Molecular Probes. Thesedextrans had a molecular weight of about 10,000 1.8 fluorophores perconjugate, and 2.2 lysines per conjugate and are sold under the tradename “Fluoro-Emerald.” Fluorescein labeled bovine serum albumin (BSA)was also obtained from Molecular Probes and had 4.5 fluors perconjugate. Various SNAFL-1/HSA conjugates were prepared as described inExample 13. Nitrocellulose membranes were obtained from Schleicher andSchuell under the trade name PROTRAN. Pore diameter was reported as 0.2microns. Capillary pore membranes made from polyester films wereobtained from Oxyphen in a variety of pore sizes. 0.1 micron and 1.0micron pore size membranes were successfully used to immobilizefluorescein dextrans. Fluorescein/dextran, fluorescein/BSA andSNAFL-1/HSA conjugates were all successfully immobilized and thefluorescent properties of the SNAFL-1/HSA conjugates were fullycharacterized as described as follows.

General Immobilization Method. SNAFL-1/HSA (2.5 SNAFL-1/HSA) on 0.1micron pore diameter Oxyphen Membrane Discs. Fluorescent HSA conjugateswith a 2.5:1 SNAFL-1:HSA offering ratio were prepared as described inExample 4 and diluted to provide concentrations of 0.05, 0.2, 1.0 and 4mg/mL in phosphate buffered saline (PBS) (pH 7.4). 5 microliter dropswere applied via a 20 microliter pipettor to the center of pre-punchedporous discs (¼ inch diameter) that were laid on a bench top. Thespotted discs were allowed to air dry (about 30 minutes) and then placedin separate desiccators overnight. The dried discs were washed inseparate Eppendorf tubes with 2×1 mL of PBS and allowed to soakovernight in 1 mL of PBS. The washed discs were stable in PBS solution(no degradation after 30 days). Alternatively the discs could bere-dried in desiccators and stored dry. The wet or dry stored discs hadcomparable fluorescent properties. The discs had fluorescent signalsthat were proportional to the concentration of labeled macromoleculeapplied to each one as measured by the fluorescence assay described inExample 9.

Example 13 The Fluorescent Properties of Immobilized Fluorophore-ProteinConjugates: SNAFL-1/HSA

Microwell assay of fluorescent macromolecular conjugates on porousmembrane discs using a fiber optic spectrometer. Fluorescent discsprepared as described in Example 8 were examined for fluorescentproperties using the Ocean Optics fiber optic spectrometer described inExample 14. The cuvette on the light source was replaced by a fiberoptic reflectance probe which had 6 excitation fibers wrapped around asingle fiber that picks up the emitted light from the sample and sendsit to the spectrometer. The reflectance probe was threaded through ahole in a 12×12×18 inch black box with a lid on the front. The probe wasclamped inside under a 1 cm square opening that allowed the tip of theprobe to be positioned under a 96-well micro well plate (clear bottomblack plate). The probe was tilted at a 30 degree angle to reducereflected light entering the probe tip. The fluorescent disc of interestwas placed in the bottom of a well and covered with 300 microliters ofthe analyte solution of interest. The excitation light source was turnedon long enough to position the disc of interest over the tip of thereflectance probe, then the shutter was closed and the plate was coveredwith another box to shield the disc from ambient light. Unless otherwisementioned, the Ocean Optics software was set to collect data with a 3000msec integration time and 3 averages. A dark spectrum was captured withthe shutter closed and used for all background subtracted readingsduring the assay. The shutter was then opened and fluorescent reading ofthe disc was started. The graphical display on the computer screen gavereal-time spectra after each 3000 msec integration time. After therequired 3 spectra were obtained (about 10 seconds) the graphicaldisplay showed only subtle changes. At this point a snapshot of thedisplayed spectrum was captured and saved to disc for future processing.The shutter was closed, and the next microwell experiment was set up.The same disc could be measured multiple times by exchanging the analytesolution in the microwells. Alternatively, different discs in differentwells could be measured by re-positioning the microwell plate over thereflectance probe.

Fluorescent loading of SNAFL-1/HSA immobilized on Oxyphen discs. Themicrowell assay described above was used to compare therelative-fluorescence of SNAFL-1/HSA on Oxyphen discs. The excitationfilters in the halogen light source were set to a wavelength of 532 nmand a “wide open” bandpass position to maximize sensitivity of theassay. Reflectance of the excitation beam back into the detector fiberwas significant, and the wavelength position of the filter was adjustedto provide a “minimum” at 620 nm where the fluorescence from the baseform of SNAFL-1/HSA is greatest. The various concentrations ofSNAFL-1/HSA described in Example 16 were examined in separate microwellsin pH 7 potassium phosphate buffer (50 mM) as described above. Thespectra showed the ability to distinguish relative fluorescenceintensity of 4, 1, 0.2 and 0.05 mg/mL membranes at pH 7. All had signalgreater than background.

Relative fluorescence intensity of various amounts of SNAFL-1/HSA(2.5:1) immobilized on porous Oxyphen discs at pH 7. The fluorescentintensity was measured at 620 nm, the fluorescent maximum of the baseform of the fluorophore. Excitation used a wide open setting on thehalogen lamp that efficiently excites both acid and base forms ofSNAFL-1. The reflected light from the source (unmodified disc) had thelowest intensity spectrum. The spectrum of the 0.05 mg/mL disc gave asmall increase in fluorescence intensity. The 0.2 mg/mL disc, 1 mg/mLdisc, and 4 mg/mL disc showed stepwise increases in fluorescenceintensity. The fluorescence spectra of two 30 day PBS soaked sample (1mg/mL) from a different batch of membranes were essentially the same andshowed that membrane loading was reproducible from batch to batch, andthat the SNAFL-1/HSA conjugates did not dissociate significantly fromthe disc surface in PBS solution.

pH Dependent fluorescence of SNAFL-1/HSA immobilized on Oxyphen discs.The 1 mg/mL SNAFL-1/HSA discs described above were examined for pHdependent response in the microwell assay. A single disc was examined inpotassium phosphate buffers of pH 4, 5, 6, 7, 8, 9, and 10. The datashowed that these membrane discs had a wide dynamic range of pHmeasurement, but had more sensitive response at pH>6. The time betweenbuffer exchanges was 5 min, and there was no significant change inspectra after additional equilibration time. This showed that theresponse time for even dramatic changes in the pH environment of theimmobilized SNAFL-1/HSA conjugates is rapid.

“Crossover assay” for fluorescence measurement of pH using SNAFL-1/HSAOxyphen discs. The microwell assay described above was used to examinethe fluorescent isosbestic properties of the discs. For this assay, theshutter assembly in Ocean Optics halogen light source (part numberHL-2000 FHSA) was removed, and two 532 nm bandpass filters (EdmundScientific) were inserted in the cavity using a special adaptor. Thisdramatically reduced the reflected background in the spectral region ofinterest (>550 nm). The data shown are for 4 mg/mL loading discsprepared with SNAFL:HSA (5:1) conjugate. The immobilized proteinconjugate showed unusual pH vs. fluorescence properties in comparison tothe solution phase data. Instead of a fluorescent isosbestic point at575 nm, there was a stepwise increase in the fluorescent intensity as pHincreased. The pH 10 spectrum showed the expected maximum at 620 nm, andcrossed the overlaid spectral curves obtained in pH 4, 6, 7 and 8buffers. These “crossover points” were used as the basis for a sensitiveassay to determine pH of the membrane environment. Three differentmembrane discs were examined using this assay format on three differentdays. The crossover points were reproducible within 2 nm.

The 4 mg/mL discs (3.6:1 SNAFL-1:HSA) showed stepwise increase in pH 10“crossover.” The crossover was at 579 nm for pH 4. Three discs/threedifferent days gave the same result ±2 nm. The crossover points were at592 nm (pH 6), 600 nm (pH 7a,b), and 611 nm (pH 8). The fluorescentmaximum at pH 10 was at 620 nm, similar to the solution phaseproperties.

Example 14 The Fluorescent Properties of Immobilized Fluorophore-ProteinConjugates: EBIO-3/HSA

Telescoping tubing insert assay of fluorescent macromolecular conjugateson porous membrane discs using a fiber optic spectrometer. Fluorescentdiscs prepared as described in Example 2 were examined to relate thefluorescent properties to the liquid phase pH using the Ocean Opticsfiber optic spectrometer described in Example 17 with the dual 532 nmfiltered (Edmund Scientific) halogen light source (part number HL-2000FHSA). A holder for a 5/32 inch membrane disc was crafted with 4 mm ODand 5 mm OD polystyrene telescoping tubing and an angled 0.015 in thickpolystyrene window. The angled window was placed so that it held themembrane disc at a 60 degree angle relative to the tubing axis. Thisallows the fiber optic probe to be placed in one end of the tubing andinterrogate the disc on the other side of the window which is contactwith a liquid of a certain pH. Buffers of known pH values were placed incontact with the telescoping tubing inserts and discs made by thespotting immobilization method in Example 2 and fluorescent emissionsrecorded with the Ocean Optics software set to collect data with a 1000msec integration time and 3 averages.

For liquids with unknown pH values, a stirred and light protected vesselcontaining 5 telescoping tubing inserts and discs made by the soakingimmobilization method in Example 2, 50 mL of buffer or plasma, and acalibrated pH electrode (ROSS electrode/Orion 720a meter) was used tostudy the pH and fluorescent response of the fluorescent discs. Drops of1 N HCl or 1 M NaOH were added to create a range of pHs from liquidsstudied. Fluorescent spectra were collected through Ocean Optics macrosin Excel set to read for 1000 msec integration time and three averages.The spectra were analyzed using the modeled bandpass filters andratiometric method in Excel to obtain calibration curves for PBS,platelet poor plasma and platelet rich plasma.

Injection molded insert PVC tube assay of fluorescent macromolecularconjugates on porous membrane discs using a custom optimizedfluorescence based pH detector. Injection molded polycarbonate partswere fashioned to fix the fluorescent discs to the fluorescence pHdetector probe as pictured in FIG. 22. Membranes were prepared asdescribed in the soaking immobilization method in Example 2 andassembled into the plastic insert. A 1 in long and 3/16 in ID PVC tubewas placed on the spike end of the insert such that 250 ul of liquid wasplaced in the tube and covered with parafilm to slow carbon dioxidedesorption. A fluorescent measurement of the first and secondwavelengths was taken and then the pH was read by a blood gas analyzer(Bayer 348). The pH of plasma samples were adjusted by acid and baseadditions as in the telescoping tubing insert assay to create the rangeof pH data.

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

1. A method for testing the sterility of a sample comprising: (a)providing a filter device having a chamber and a filter configured tocollect culturable organisms; (b) filtering a sample through the filtersuch that any collected culturable organisms are retained in thechamber; (c) adding a culture medium to the chamber; (d) irradiating afluorescent species immobilized on a substrate with excitation lightemanating from a probe physically isolated from the fluorescent speciesimmobilized on the substrate, wherein the fluorescent speciesimmobilized on the substrate is in liquid communication with the culturemedium, wherein the excitation light has a wavelength sufficient toeffect fluorescent emission from the fluorescent species, and whereinthe fluorescent species exhibits a first emission intensity at a firstemission wavelength and a second emission intensity at a second emissionwavelength, the ratio of the first and second emission intensities beingdependent on pH; (e) measuring the first and second emission intensitiesto determine the pH of the culture medium; and (f) inferring from thedetermined pH of the culture medium the sterility of the sample.
 2. Themethod of claim 1, wherein the filter comprises a membrane having a poresize of about 45 μm.
 3. The method of claim 1, wherein the probe isphysically isolated from the fluorescent species immobilized on thesubstrate by a window transparent to the excitation light and thefluorescent emission.
 4. The method of claim 1, wherein the probecomprises one or more optical fibers.
 5. The method of claim 1, whereinthe fluorescent species is selected from the group consisting of anaphthofluorescein compound and a seminaphthorhodamine compound.
 6. Themethod of claim 1, wherein the fluorescent species is anaphthofluorescein compound selected from the group consisting of aseminaphthofluorescein compound and a carboxynaphthofluoresceincompound.
 7. The method of claim 1, wherein the fluorescent species is aseminaphthofluorescein compound selected from the group consisting of5′(and6′)-carboxy-3,10-dihydroxy-spiro[7H-benzo[c]xanthene-7,1′(3′H)-isobenzofuran]-3′-oneand2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoicacid.
 8. The method of claim 1, wherein the fluorescent speciescomprises a conjugate of a fluorescent compound and a macromolecule. 9.The method of claim 8, wherein the macromolecule is an albumin.
 10. Themethod of claim 1, wherein the fluorescent species comprises aseminaphthofluorescein/human serum albumin conjugate.
 11. The method ofclaim 1, wherein the sample comprises blood or a blood product.
 12. Amethod for testing the sterility of a sample, comprising: (a) providinga filter device having a chamber and a filter configured to collectculturable organisms; (b) filtering a sample through the filter suchthat any collected culturable organisms are retained in the chamber; (c)adding a culture medium to the chamber; (d) irradiating a fluorescentspecies in communication with the culture medium with excitation lighthaving a wavelength sufficient to effect fluorescent emission from thefluorescent species, wherein the fluorescent species exhibits a firstemission intensity at a first emission wavelength and a second emissionintensity at a second emission wavelength, the ratio of the first andsecond emission intensities being dependent on pH, and wherein thefluorescent species comprises a 2-halo seminaphthofluorescein; (e)measuring the first and second emission intensities to determine the pHof the culture medium; and (f) inferring from the determined pH of theculture medium the sterility of the sample.
 13. The method of claim 12,wherein the 2-halo seminaphthofluorescein is a 2-chloroseminaphthofluorescein.
 14. The method of claim 12, wherein the 2-haloseminaphthofluorescein is2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoicacid.
 15. The method of claim 12, wherein the fluorescent speciescomprises a conjugate of a 2-halo seminaphthofluorescein and amacromolecule.
 16. The method of claim 15, wherein the macromolecule isan albumin.
 17. The method of claim 15, wherein the macromolecule is ahuman serum albumin.
 18. The method of claim 15, wherein the conjugateis a2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoicacid/human serum albumin conjugate.
 19. The method of claim 12, whereinthe sample comprises blood or a blood product.
 20. An apparatus fortesting the sterility of a sample liquid comprising: (a) a chamberhaving an inlet and an outlet; (b) a filter disposed at the chamberoutlet, the filter adapted to collect culturable organisms from a liquidpassed through the filter; and (c) a port assembly having a distal endextending into the chamber, the distal end comprising a fluorescentspecies immobilized on a substrate, wherein the fluorescent species isin fluid communication with an interior of the chamber, wherein thefluorescent species comprises a material that, when irradiated with anexcitation light having a wavelength sufficient to effect fluorescentemission from the fluorescent species, exhibits a first emissionintensity at a first emission wavelength and a second emission intensityat a second emission wavelength, the ratio of the first and secondemission intensities being dependant on pH, and wherein the portassembly further comprises a housing for receiving a probe.
 21. Theapparatus of claim 20, wherein the filter comprises a membrane having apore size of about 45 μm.
 22. The apparatus of claim 20, wherein thehousing comprises an open first end and a second end terminating with awindow.
 23. The apparatus of claim 22, wherein the port assemblycomprises a tip member reversibly connectable to the housing second end,wherein the tip member is adapted to receive liquid from a sample to betested.
 24. The apparatus of claim 20, further comprising: (a) a lightsource for exciting the fluorescent species; (b) a first emissiondetector for measuring the first emission intensity; (c) a secondemission detector for measuring the second emission intensity; (d) anexcitation lightguide for transmitting excitation light from the lightsource to the fluorescent species, wherein the excitation lightguidecomprises a first terminus proximate to the light source and a secondterminus distal to the light source; (e) a first emission lightguide fortransmitting emission from the fluorescent species to the first emissiondetector, wherein the first emission lightguide comprises a firstterminus proximate to the first emission detector and a second terminus;(f) a second emission lightguide for transmitting emission from thefluorescent species to the second emission detector, wherein the secondemission lightguide comprises a first terminus proximate to the secondemission detector and a second terminus; and (g) a probe housing,wherein the probe housing houses the second termini of the excitationlightguide, first emission lightguide, and second emission lightguide.25. The apparatus of claim 24, wherein the light source is alight-emitting diode.
 26. The apparatus of claim 24, wherein the firstand second emission detectors are photodiodes.
 27. The apparatus ofclaim 24, wherein the excitation lightguide, the first emissionlightguide, and the second emission lightguide are optical fibers. 28.The apparatus of claim 22, wherein the port assembly comprises a taperedtube terminating with the window.
 29. The apparatus of claim 20, whereinthe fluorescent species comprises a seminaphthofluorescein/human serumalbumin conjugate.
 30. An apparatus for testing the sterility of asample liquid comprising: (a) a chamber having an inlet and an outlet;(b) a filter disposed at the chamber outlet, the filter adapted tocollect culturable organisms from a liquid passed through the filter,the filter having a fluorescent species immobilized thereon, wherein thefluorescent species is in fluid communication with an interior of thechamber, wherein the fluorescent species comprises a material that, whenirradiated with an excitation light having a wavelength sufficient toeffect fluorescent emission from the fluorescent species, exhibits afirst emission intensity at a first emission wavelength and a secondemission intensity at a second emission wavelength, the ratio of thefirst and second emission intensities being dependant on pH; and (c) aport assembly having a distal end adjacent to the filter, the portassembly further comprising a housing for receiving a probe.
 31. Theapparatus of claim 30, wherein the filter comprises a membrane having apore size of about 45 μm.
 32. The apparatus of claim 30, wherein thehousing comprises an open first end and a second end terminating with awindow.
 33. The apparatus of claim 30, further comprising: (a) a lightsource for exciting the fluorescent species; (b) a first emissiondetector for measuring the first emission intensity; (c) a secondemission detector for measuring the second emission intensity; (d) anexcitation lightguide for transmitting excitation light from the lightsource to the fluorescent species, wherein the excitation lightguidecomprises a first terminus proximate to the light source and a secondterminus distal to the light source; (e) a first emission lightguide fortransmitting emission from the fluorescent species to the first emissiondetector, wherein the first emission lightguide comprises a firstterminus proximate to the first emission detector and a second terminus;(f) a second emission lightguide for transmitting emission from thefluorescent species to the second emission detector, wherein the secondemission lightguide comprises a first terminus proximate to the secondemission detector and a second terminus; and (g) a probe housing,wherein the probe housing houses the second termini of the excitationlightguide, first emission lightguide, and second emission lightguide.34. The apparatus of claim 33, wherein the light source is alight-emitting diode.
 35. The apparatus of claim 33, wherein the firstand second emission detectors are photodiodes.
 36. The apparatus ofclaim 33, wherein the excitation lightguide, the first emissionlightguide, and the second emission lightguide are optical fibers. 37.The apparatus of claim 32, wherein the port assembly comprises a taperedtube terminating with the window.
 38. The apparatus of claim 30, whereinthe fluorescent species comprises a seminaphthofluorescein/human serumalbumin conjugate.
 39. A system for testing the sterility of a liquidsample, comprising: (a) a plurality of stations, each station configuredto receive a culture assembly; (b) a plurality of culture assemblies,each culture assembly comprising: (i) a chamber having an inlet and anoutlet; (ii) a filter disposed at the chamber outlet, the filter adaptedto collect culturable organisms from a liquid passed through the filter;(iii) a port assembly having a distal end extending into the chamber,the distal end comprising a fluorescent species immobilized on asubstrate, wherein the fluorescent species is in fluid communicationwith an interior of the chamber, wherein the fluorescent speciescomprises a material that, when irradiated with an excitation lighthaving a wavelength sufficient to effect fluorescent emission from thefluorescent species, exhibits a first emission intensity at a firstemission wavelength and a second emission intensity at a second emissionwavelength, the ratio of the first and second emission intensities beingdepending on pH; and wherein the port assembly further comprises a probehousing.
 40. The system of claim 39, wherein each culture assemblyfurther comprises: (a) a light source for exciting the fluorescentspecies; (b) a first emission detector for measuring the first emissionintensity; (c) a second emission detector for measuring the secondemission intensity; (d) an excitation lightguide for transmittingexcitation light from the light source to the fluorescent species,wherein the excitation lightguide comprises a first terminus proximateto the light source and a second terminus distal to the light source;(e) a first emission lightguide for transmitting emission from thefluorescent species to the first emission detector, wherein the firstemission lightguide comprises a first terminus proximate to the firstemission detector and a second terminus; (f) a second emissionlightguide for transmitting emission from the fluorescent species to thesecond emission detector, wherein the second emission lightguidecomprises a first terminus proximate to the second emission detector anda second terminus; and (g) a probe housing, wherein the probe housinghouses the second termini of the excitation lightguide, first emissionlightguide, and second emission lightguide.
 41. A system for testing thesterility of a liquid sample, comprising: (a) a plurality of stations,each station configured to receive a culture assembly; (b) a pluralityof culture assemblies, each culture assembly comprising: (i) a chamberhaving an inlet and an outlet; (ii) a filter disposed at the chamberoutlet, the filter adapted to collect culturable organisms from a liquidpassed through the filter, the filter having a fluorescent speciesimmobilized thereon, wherein the fluorescent species is in fluidcommunication with an interior of the chamber, wherein the fluorescentspecies comprises a material that, when irradiated with an excitationlight having a wavelength sufficient to effect fluorescent emission fromthe fluorescent species, exhibits a first emission intensity at a firstemission wavelength and a second emission intensity at a second emissionwavelength, the ratio of the first and second emission intensities beingdependant on pH; and (iii) a port assembly having a distal end adjacentto the filter, the port assembly further comprising a probe housing. 42.The system of claim 41, wherein each culture assembly further comprises:(a) a light source for exciting the fluorescent species; (b) a firstemission detector for measuring the first emission intensity; (c) asecond emission detector for measuring the second emission intensity;(d) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the excitationlightguide comprises a first terminus proximate to the light source anda second terminus distal to the light source; (e) a first emissionlightguide for transmitting emission from the fluorescent species to thefirst emission detector, wherein the first emission lightguide comprisesa first terminus proximate to the first emission detector and a secondterminus; (f) a second emission lightguide for transmitting emissionfrom the fluorescent species to the second emission detector, whereinthe second emission lightguide comprises a first terminus proximate tothe second emission detector and a second terminus; and (g) a probehousing, wherein the probe housing houses the second termini of theexcitation lightguide, first emission lightguide, and second emissionlightguide.